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PURIFICATION OF A EUKARYOTIC 
RNA POLYMERASE II THAT SYNTHESIZES 
POLYADENYLIC ACID 



By 

ROBERT HENRY BENSON 



A DISSERTATION PRESENTED TO THE GRADUATE 
COUNCIL OF THE UNIVERSITY OF FLORIDA IN PARTIAL 
FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF 

DOCTOR OF PHILOSOPHY 



UNIVERSITY OF FLORIDA 
1S72 



This dissertation is dedicated to my parents, 
Mary Ann L. and Henry E. Benson, for their end- 
less encouragement during my 25 years of 
education . 



. ,,. ,.,^.,= ACKNOWLEDGMENTS 

I would like to thank Dr. Rusty J. Mans for all the 
time and effort he has put forth on my behalf, both 
scientifically and personally. At first I didn't know 
quite ,i how A.to .d-.eal . with his enthusiastic approach to prob- 
lems, but gradually I learned. I would like to thank 
Dr. George E. 'Safford for introducing me to microbiology 
and teaching me that research could be both enjoyable and 
challenging. f would like to thank Dr. Ira Rosen for 
introducing me to ^genetics and transcription and I would 
like to thank Dr. Daniel Billen for introducing me to 
Dr. Rusty J. Mans. As usual, there are all the other 
people and I would like to thank them too. I would like 
to thank Muriel Reddish who knows everything that a 
graduate student ever needs to know about the department. 
My thanks to Claudia Alverez who shares everyone's 
laboratory frustrations, especially those of Dr. Rusty J. 
Mans. My thanks to Sharon Bryant, Bob Brooks, Gary Benson 
Carl Smith and Norm Huff for keeping me company in our 
one-windowed laboratory. I would really like to thank the 
N. I. H. which has kept me lean but alive during the last 
five years. To my roommates, Barbara, Joe and Chantal , 
"thanks, and whose turn is it to do the dishes?" And in 



conclusion, I would like to thank John Henry Colson and 
Bernardine, for keeping me company during all my nights 
in the laboratory repeating experiments and typing endless 
drafts of this dissertation. 



TABLE OF CONTENTS 



Acknowledgments iii 

List of Tables vi 

List of Figures vii 

Key to Abbreviations ix 

Abstract xi 

Introduction 1 

Literature Review 4 

Methods and Materials. . . . 21 

Results 31 

Discussion 86 

Conclusion 99 

References . 100 

Appendix A 105' 

Appendix B 109 

Biograpbxical Sketch 112 



LIST OF TABLES 



1. STEPS IN KNA POLYMERASE PURIFICATION 32 

2. STORAGE AND FREEZE-THAW LABILITY OF RNA 
POLYMERASE 40 

3. SALT PRECIPITATION OF RNA POLYMERASE 42 

4. ASSAY REQUIREMENTS OF RNA POLYMERASE 44 

5. NEAREST NEIGHBOR FREQUENCY ANALYSIS OF RNA 

PRODUCT SYNTHESIZED WITH DENATURED DNA. 59 

6. AMP AND UMP' INCORPORATION BY RNA POLYMERASE ... .65 

7. INHIBITION OF POLY ADENYLIC ACID SYNTHESIS BY 
NUCLEOTIDES 67 

8. EFFECT OF DELAYED ADDITION OF NTPs ON POLYADENYLIC 
ACID SYNTHESIS. 68 

9. EFFECT OF HEATING' AND SDS ON RNA POLYMERASE 
PRODUCTS, 71 

10. NEAREST NEIGHBOR FREQUENCY ANALYSIS OF POLY- 
ADENYLIC ACID PRODUCT .72 

11. AVERAGE CHAIN LENGTH OF POLYADENYLIC ACID 73 

12. INHIBITOR SENSITIVITY OF RNA POLYMERASE 76 

13. COMPARISON OF CALF THYMUS AND MAIZE DNA AS 
TEMPLATES FOR RNA POLYMERASE. 82 

14. RNA POLYMERASE ACTIVITY WITH SYNTHETIC TEMPLATES. .84 



vi 



LIST OF FIGURES 



1.. PURIFICATION PROCEDURE FOR RNA POLYMERASE 22 

2.. DEAE-CELLULOSE GRADIENT ELUTION ASSEMBLE 25 

3. DEAE-CELLULOSE ELUTION PROFILE' 34 

4. RNA POLYMERASE ACTIVITY IN DEAE-CELLULOSE 
FRACTIONS 35 

5. POLYACRYLAMIDE GEL ELECTROPHORESIS OF NATIVE RNA 
POLYMERASE 37 

6. POLYACRYLAMIDE GEL ELECTROPHORESIS OF DENATURED 

RNA POLYMERASE 38 

7. DNA TITRATIONS WITH MAGNESIUM AS COFACTOR 46 

8. DNA TITRATIONS WITH MANGANESE AS COFACTOR 47 

9. MAGNESIUM TITRATION WITH NATIVE AND DENATURED DNA. 49 

10. ■ MANGANESE TITRATION WITH NATIVE AND DENATURED DNA. 50 

11. NTP TITRATION OF, RNA POLYMERASE.. 51 

12. ATP TITRATION OF RNA POLYMERASE 53 

13. LINEWEA¥ER-BURKE PLOTS OF ATP TITRATIONS 54 

14. AMMONIUM SULFATE TITRATION OF RNA POLYMERASE ... 55 

15. RNA POLYMERASE ACTIVITY AS A FUNCTION OF ENZYME 
CONCENTRATION. •, 57 

16. RATE OF AMP INCORPORATION AS A FUNCTION OF TIME. . 58 

17. DNA TITRATIONS WITH MAGNESIUM IN THE ABSENCE OF 

NTPs 62 

18. DNA TITRATIONS WITH MANGANESE IN THE ABSENCE OF 

NTPS 63 

vii 



LIST OF FIGURES (continued) 



19. NTP TITRATION WITH DENATURED DNA 66 

20. POLYADENYLIC ACID SYNTHESIS AS A FUNCTION OF 

ENZYME CONCENTRATION 70 

21. ALPHA-AMANITIN TITRATION OF RNA POLYMERASE. ... 78 

22. ACTINOMYCIN D TITRATION OF RNA POLYMERASE .... 79 

23. RIBONUCLEASE SENSITIVITY OF DENATURED DNA-DEPENDENT 
PRODUCTS 81 

24. AMP INCORPORATION AS A FUNCTION OF ATP SPECIFIC 
ACTIVITY 106 

25. RATE OF AMP INCORPORATION AS A FUNCTION OF ATP 
CONCENTRATION 108 

26. A MODEL FOR POLYADENYLIC ACID INITIATION OF 
TRANSCRIPTION 110 



viii 



KEY TO ABBREVIATIONS 



^2S0 absorbancy at 260 nm 

ATP adenosine triphosphate 

AMP adenosine monophosphate 

BSA bovine serum albumin 

CAP catabolite gene-activator protein 

cpm counts per minute 

CTP cytidine triphosphate 

. d dalton 

DMSG dimethyl sulfoxide 

dpm disintegrations per minute 

DEAE diethylaminoethane 

DNA deoxyribonucleic acid 

GTP guanosine triphosphate 

■ g gravity • 

h hour 

HrRNA • heterogeneous nuclear ribonucleic acid 

NTPS - GTP, CTP, UTP 

POPOP l,4-bis-,[2- (4-methyl-5-phenyloxazolyl) ]- 

benzene 

PPG 2 , 5-diphenyloxazole 

poly (A) polyadenylic acid 

polyCdAT) alternating copolymers of deoxyadenylic 

and deoxythymidylic acid 

poly(U) polyuridylic acid 

poly{dAdT) homopolymers of deoxyadenylic and 

deoxythymidylic acid- 

ix 



KEY TO ABBREVIATIONS (continued) 



RNA ribonucleic acid 

rRNA ribosomal ribonucleic acid 

S sedimentation coefficient 

SSC standard saline citrate 

SDS sodium dodecyl sulfate 

TCA trichloroacetic acid 

tris tris (hydroxymethyl) aminomethane 

tJRNA transfer ribonucleic acid 

UTP uridine triphosphate 

UMP uridine monophosphate 

50% ASP 50% ammonium sulfate precipitate 



X 



Abstract of Dissertation Presented to the Graduate Council 
of the .University of Florida in Partial Fulfillment of the 
Requirements for the Degree of Doctor of Philosophy 



PURIFICATION OF A EUKARYOTIC RNA POLYMERASE II 
THAT SYNTHESIZES POLYADENYLIC ACID 

By- 

Robert Henry Benson 
December, 1972 

Chairman: Rusty J. Mans, Ph.D. 

Major Department: Immunology and Medical Microbiology 

The in vitro catalytic activities of DNA-dependent 
RNA polymerase II, isolated and purified from rapidly 
growing 5-day-old maize seedlings (WF9 x Bear 38, waxy), 
have been examined. An enzyme purification procedure is 
described utilizing homogenization, differential centri- 
fugation, salt precipitation and ion exchange chromoto- 
graphy that resulted in 1,000-fold purification with 
70 percent recovery in 6 to 10 hours. The enzyme had a 
specific activity greater than 100 nmoles AMP/mg/20 min. 
at 30°, and contained one major band and several minor 
bands on polyacrylamide gel electrophoresis. Incorpora- 
tion of AMP into acid-insoluble material required DNA, 

4 nucleosidetriphosphates : GTP , CTP, UTP, ATP (NTPs) , 

2+ 2+ 

and metal, either Mn or Mg . Alkaline hydrolysis of 

xi 



product, synthesized with [a--^^'P]AT'P , NTPs, denatured DNA 
2+ 

and Mn , resulted in these nearest neighbor frequencies: 
CpA, 0.25; ApA, 0.44; GpA, 0.18; UpA, 0.13. The require- 
ments for incorporation of a labeled nucleotide into acid- 
insoluble material and the nearest neighbor frequency 
indicate that RNA was synthesized. In the presence of 
labeled ATP alone, the ApA frequency of alkaline-hydrolyzed 
product was 0.90, and the AMP/adenosine ration was greater 
than 100, indicating polyadenylic acid [poly (A) ] was syn- 
thesized. Since UTP was incorporated in the presence but 
not in the absence of NTPs, no poly(U) was synthesized, 
suggesting that poly (A) may be the only homopolymer 
accxamulated. Poly (A) synthesis was 90 percent inhibited 
by 0.025 itiM NTPs, whereas both RNA and poly (A) were syn- 
thesized with 1.0 mM (or greater) NTPs, suggesting the 
mode of poly (A) synthesis differs as a function of the nu- 
cleotides present, but all products synthesized, were acid- 
insoluble after heating (100°) for 5 min. in 1 percent SDS , 
indicating that product-protein and product-DNA complexes 
were not responsible for acid-insolubility. RNA and 
poly (A) synthesis was inhibited by 1 pM a-amanitin, 
indicating that both products were synthesized by RNA 
polymerase II. Poly (A) synthesis was supported by poly(dAdT), 
but not by poly(dAT), suggesting a poly{dT) template was 

utilized for poly (A) synthesis. Actinomycin D (7 yM) 
inhibited RNA synthesis 87 percent on denatured calf thymus 

xii 



DNA, but stimulated poly CA) synthesis 28 percent, again 
suggesting poly (dT) regions of DNA were utilized as a 
template for poly (A) synthesis. Neither RNA nor poly (A) 
synthesis was inhibited by cordycepin (0.26 mK) or 
rifampicin (50 yg/ml) , therefore eliminating the presence 
of maize NTP: exotransf erase and bacterial RNA polymerase 
-activity. Since early product (accumulated at a high rate 
of AMP incorporation) was resistant to pancreatic ribo- 
nuclease, whereas, late product (accumulated at a lower 
rate of AMP accumulation) was sensitive to RNase digestion 
poly (A) synthesis apparently preceded ^^NA synthesis. A 
model is presented which requires that poly (A) synthesis 
..precede RNA synthesis in the same enzyme-template complex 
during the initiation of transcription in eukaryotes. 



xiii 



INTRODUCTION 



Macromolecular nucleic acid metabolism depends upon 
two classes of enzymes: the polymerases which assemble 
the polymers from the nucleoside or deoxynucleoside tri- 
phosphates, and the nucleases which break down the polymers. 
Among the polymerases there are four types which are known 
to utilize a nucleic acid as a template to synthesize a 
new polymer. These are DNA-dependent DNA polymerase 
(EC 2.7.7.7), DNA-dependent RNA polymerase (EC 2.7.7.6), 
RNA-dependent RNA polymerase and RNA-dependent DNA poly- 
merase. - Of primary importance in phenotypic expression of 
genetic information are the DNA-dependent RNA polymerases. 
These enzymes are responsible for the synthesis of messenger 
RNA, the template for sequencing amino acids, and for the 
synthesis of ribosomal and transfer RNAs , needed for trans- 
lation of the messenger RNA into protein. Control of the 
synthesis of these RNAs seems to be primarily at the level 
of initiation of transcription. 

Transcription may be studied in vivo or in vitro . 
The in vitro approach to transcription requires a purified 
RNA polymerase. Once the enzyme is purified, specific 
properties can ten be determined, such as the assay 



1 



a 



requirements and product characteristics. Following 
this initial evaluation, transcriptional control functions, 
such as template specificity or the presence or absence 
of initiation and termination factors, can be determined. 

The DNA-dependent RNA polymerases all require a 
DNA template, the four nucleoside triphosphates: ATP, 
UTP, GTP and CTP , and metal during the synthesis of RNA 
and concomitant liberation of pyrophosphate. The RNA 
polymer is synthesized in the 5' to 3' direction. The 
new nucleotide is linked via a phosphodiester linkage to 
the 3' hydroxyl of the growing RNA chain. As the RNA is 
synthesized in the 5' to 3' direction, the template DNA 
is transcribed in the 3' to 5' direction. RNA polymerase 
activity can be evaluated separately in four operational 
steps: binding, initiation, elongation and termination. 
Step one is the binding of the RNA polymerase to the DNA 
template. Step two is the initiation of an RNA chain 
via synthesis of the first phosphodiester linkage. Step 
three is the elongation of the new RNA chain. Step four 
is the termination of RNA synthesis. Release of the RNA 
chain may occur immediately or it may be delayed. 

Experimental evaluation of the initial steps, 
binding to the DNA template and initiation of RNA syn- 
thesis, requires an enzyme of high specific activity. 
This high activity is required to experimentally detect 



3 



the initial synthetic activity of the RNA polymerase 
and to minimize the influence of any other enzymatic 
activities present. In addition, experimental manipu- 
lation of the enzyme during purification should be kept 
minimal, to reduce the probability of enzyme degeneration 
and, therefore, to preserve in vivo transcriptional 
activity. The experimental approach in this study was 
to purify the 5ttJA polymerase from maize seedlings, to 
determine its requirements for product accumulation and 
to identify the products. RNA polymerase was purified 
by centrif ugation, {NH^)2S0_^ precipitation and ion ex- 
change column chromotography in that order. From its 
assay requireisents and inhibitor sensitivities it was 
identified as HNA polymerase type II. In addition to its 
known catalysis of RNA synthesis, this eukaryotic RNA 
polymerase catalyes polyadenylic acid synthesis . The 
polymerase products were characterized by sensitivity to 
heat, SDS and pancreatic ribonuclease , and by nearest 
neighbor frequency analysis. The possible involvement of 
poly (A) synthesis as a mechanism of initiation of tran- 
scription is suggested and discussed. 




LITERATURE REVIEW 

Approaches to Transcription 

Guides to Literature 

There are several avenues into the literature of 
transcription; for curxent research, the symposia (1,2,3) 
and reviews (4,5,6) are best, whereas for a more historical 
perspective, there are collected papers (7) and introductory 
texts (8,9,10) . • • < 

Systems Studied 

Essentially every level of life from the smallest 
viruses to the largest eukaryotes has had, or is having, 
its transcriptional processes studied. The basic enzymology 
has been done in bacterial systems, principally Escherichia 
coli and Bacillus subtilis and their phages (11,12). 
Although RNA polymerase was first detected in a eukaryote 
by Weiss in 1960 (13), the bacterial viruses and their 
nucleic acids proved most useful in elucidating transcription 
in prokaryotic systems (11,14). The major difficulties en- 
countered in studying the eukaryotic systems were the initial 
low activity of the eukaryotic RNA polymerases and the dif- 
ficulty in getting the enzyme DNA-dependent (15) . These 

' 4 ■ 



i 



problems have been solved and the study of in vitro 
transcription in eukaryotes is rapidly advancing. 

Transcription as a subject may be broken into two 
areas of investigation: first, the prokaryotes and their 
viruses that have served as model systems due to the ease 
of acquiring enzyme and defined DNA templates; second, the 
eukaryotes that have complex' chromosomes and multiple 
polYmerase. systems producing the intricate specialized 
tissues of plants and animals. The bacterial systems will 
be illustrated by the E. coli and B. subtilis systems and 
the eukaryotes by the calf thymus and maize systems. 

Bacterial Transcription 

Significant Concepts 

There -are extensive reviews of transcription available 
(1,2,4,5,6). I would like to draw from these systems to 
illTastrate four concepts which have recently been uncovered 
in bacterial transcription. , . • 

, ■ 1. KNA polymerase and associated factors have 
the ability to initiate and , terminate tran- 
■ - scription at specific sequences on the 

chromosome. 

2, Alteration of the RNA polymerase, either 
through viral infection or sporulation, 
results in a change in the transcription 
specificity of the RNA polymerase. 



3. RNA polymerase may be the site of hormone 
action via specific protein factors such 
,, . as CAP (catabolite gene-activator protein) 

which are influenced by hormone-controlled 
cyclic AMP. 

; 4. Alteration of the RNA polymerase transcription 

specificity can result in irreversible 
_ , ■ . changes in cell phenotype, closely 

resembling differentiation in eukaryotes. 

These four concepts illustrate the potential importance of 
a better understanding of the RNA polymerases in both pro- 
karyotes and in eukaryotes. 

E. Coli RNA Polymerase and Sigma Factor ' ' 

I- ^Qli polymerase contains four types of subunits: 

B' (155-165,000 d) , 6 (145-155,000 d) , a (85-95,000 d) , and 
a (39-41,000 d) . A fifth subunit, oj (10,000 d) , is some- 
times found with the RNA polymerase although it is not 
required for RNA polymerase function (4) . Together these 
subunits are arranged as holenzyme ( 3 ' Ba a), which retains 

2 

the ability to asymmetrically transcribe T4 DNA, or as core 
enzyme (g'Ba^), which cannot asymmetrically transcribe T4 
DNA (11) . The presence or absence of sigma (a) determines 
the initiation specificity of the RNA polymerase on T4 DNA. 
Another factor, rho (p ),. although not bound to the RNA poly- 
merase, when present with the enzyme resulted in one type 
of specific termination of RNA synthesis (15) . Two other 
sites also resulted in chain termination. The RNA poly- 
merase, without rho, also recognized sequences in the DNA 
transcribed as UAA (ochre) and UAG (amber) and terminated 



•RKA chains (15). . • • 

In response to infection by coliphage T4, E. coli 
RM& polymerase transcribed early T4 RNA. After production 
of the gene 55 protein, translated from the early RNA, the 
synthesis of delayed early RNA was specifically initiated 
(16) . Travers (17) isolated the protein and called it a 
T4 sigma-like factor which caused specific asymmetric 
initiation of T4 DNA at the site of delayed early RNA. This 
isolation of a factor wfiich directed delayed early RNA 
synthesis demonstrated for the first time that RNA poly- 
merase could acquire a new initiation specificity (16,17). 
One minute after infection by phage T4, the host RNA poly- 
merase no longer synthesized early T4 RNA (18) . This 
change was apparently caused by alteration of the a sub- 
unit of the host RNA polymerase through adenylation with 
5'AMP (19). Therefore, viral infection initiated changes 
in the transcription machinery which caused a specific 
alteration of the initiation sites for RNA synthesis. 
These changes included alteration of the existing host RNA 
polymerase and the synthesis of a viral protein to replace 
the host sigma factor. 

E. Coli RNA Polymerase and CAP 

RNA polymerase control in coli illustrates the 
possible importance of transcription as a mechanism for 
tonnone action. Cyclic AMP, together with CAP, was required 
for maximum expression of the lactose and other inducible 



i 



■operons {20,21). CAP and cyclic AMP were bound to the 
lactose repressor protein and catalyzed its dissociation 
from the operator gene (21) . The results with CAP raised the 
possibility that transcription of many prokaryotic and 
eukaryotic genes requires the action of an additional 
positive control element. The requirement for cyclic AMP 
linked transcriptional control to hormone action (22) . 

B. Subtilis RMA Polymerase and Sporulation 

The sporulation of B. subtilis resulted in changes in 
the template specificity of the RNA polymerase. These 
changes were associated with a decrease in transcription 
of vegetative genes and the expression of new sporulation 
specific genes (23,24) . , The sporulating cell contained 
an RNA polymerase with a 3 subunit alteration such that it 
could not utilize vegetative sigma factor (25) . This ir- 
reversible change in cell phenotype resembled differentia- 
tion, since once sporulation began, the cell was committed 
and could not return to vegetative growth except through the 
sporulation stage. .' . 

Eukaryotic Transcription 

General Background 

While eukaryotic RjSIA polymerase activity was detected 
as early as 1950 by Weiss (13) , initial progress at 



purif-ic.atir'n7*.w.o!^s - veKj,'- slow. This was a . result of the 
initliaJ..., I om? :::a^ytl.5?,<-t:>Y ;of the RNA polymerase in eukaryotic 
tissues, of tiiS'i-iSif^i-c.ulty encountered in freeing the enzyme 
of contaminating ''DNA and of the instability of the enzyme 
during purification (26) . These problems were overcome and 
soluble DNA-dependent RNA polymerases were purified from 
eukaryotic tissues (27,28,29,30). In 1970, Roeder and 
Rutter (31) first detected the presence of two types of 
RNA polymerases..,.''- T;ype I was a nucleolar RNA polymerase, 
insensitive to a-amanitin, that synthesized a product which 
competitively -.riybridized with r-RNA but not with Hn-RNA. 
Type II was a nuE-.l,f?oplasmic , a-amanitin sensitive RNA poly- 
merase that synthesized a product that competitively 
hybridized with Hn-RNA but not with r-RNA (32) . This was 
the first time that specific RNA polymerases were shown 
to be localized inside a eukaryotic cell and to be re- 
sponsible for specific classes of eukaryotic RNA. Since 
then it has been shown that the nucleus, the nucleolus, 
and the cellular organelles have unique transcriptional 
systems (15) . 

Eukaryotes have a complex chromosomal structure 
involving nucleic acids, histones, and acidic proteins. 
The relationship betv/een the histones and differentiation 
is. uncertain, al-though histones are believed to be inti- 
mately involved -in gene selection (33). There are two 
major approaches to how this interaction might occur. 



First, transcription may be activated by removal of his tones, 
somewhat analogous to removal of the repressor on the lactose 
operon. Second, KNA polymerase and specific initiation 
factors initiate transcription by opening the genes for 
transcription, with histone removal occurring as the RNA 
polymerase precedes (15) . Accordingly, either the histones 
or the RNA polymerases may function to control the specificity 
of transcription, or a combination of both systems. 

Acidic nuclear proteins are of interest to transcription 
because of their physical properties and location (34) . 
since the RNA polymerases are themselves acidic nuclear 
protein complexes, some of the acidic proteins may be sub- 
units of the RNA polymerases or of the other polymerases. 
The acidic nuclear proteins may contain factors that function 
along with the histones for gene selection and control of 
transcription. 

Transcription Products 

There are at least three classes of RNA produced in 
all cells: rapidly labeled RNA including HnRNA and mRNA; 
stable RNA or GC-rich RNA which is rRNA; and soluble or , 
tRNA. These three classes of RNAs constitute the bulk of 
the transcriptional products in all cells. Following 
transcription, the RNA is often processed by specific 
systems which selectively degrade the gene product into 
the functionally active form. The processing of ribosomal 



11 



RNA Ls the i?es.t studied. The ribosomal genes are 
seq.ueB;fce-red- in the nucleolus and are transcribed as a unit 
into- -a s:mg.le -40-45 S precursor RNA molecule. This RNA is 
■pxocessed by a series of post-transcriptional cleavage steps 
to give 18 S and 25 S RNAs found in mature amphibian oocyte 
ribosomes (35) . Heterogeneous RNA is found in the nucleo- 
plasm and has a DNA-like base composition. A large fraction 
of this rapidly labeled RNA never leaves the nucleus and 
may be involved with regulation at the level of transcription 
(36). Both rRNA and tRNA are stable and constitute the 
bulk of the RNA contained in a cell at any one time, while 
most of the- rRNA rapidly turns over. 

RNA Polymerases 
Types 

Type I RNA polymerase is localized in the nucleolus 
and synthesizes GC-rich RNA that competitively hybridizes 
with ribosomal RNA ^32) . Its polymerizing activity is 
resistant to ct-amanitin, it is highly sensitive to actinomycin 
D and it is refractory to rifampicin (32) . On DEAE-sephadex 
it is the first RNA polymerase eluted by a linear ammonium 
sulfate gradient, generally around 0.2 M (NH^)2S0^. The 
type I RNA polymerase has a preference for magnesium and 
low salt concentrations (0.04-0.07 M {NH^)2S0^). The type I 
RNA polymerase is very unstEible and therefore difficult to 
purify (37) . 



12 



The type IT RNA polymerase is a nucleoplasmic RNA 
polymerase that synthesizes a product which competitively 
hybridizes with HnRNA. (32) . Its activity is sensitive 
to a-amanitin and it is the second RNA polymerase eluted 
by a linear (NH^)2S0^ gradient from DEAE-sephadex , generally 
around 0.3 M salt. The type II enzyme exhibits more 
activity with manganese, rather than with magnesium, and 
is most active at high salt concentrations (0.9-0.12 M 
(NH^)2S0^) (32). It is more stable than the type I RNA 
polymerase, but is inactivated easily, particularly during 
salt precipitation. . 

Type. Ill RNA polymerase is a nucleoplasmic RNA 
polymerase, eluted third on DEAE-sephadex chromotograph, 
generally around 0.35.M (NH^)2S0^ (32). The small amount 
of type III enzyme activity present in extracts is often 
undetected. The type III RNA polymerase is resistant to 
a-amanitin, prefers manganese and has a broad salt optimum 
(0-0.2 M (NH^)2S0^) (32). It is the least studied of the 
nuclear RNA polymerases and it has been proposed that it may 
synthesize tRNA (32) . • 

The type IV RNA polymerase, refers to RNA polymerase 
activity detected in cellular organelles, specifically 
chloroplasts and mitochondria. Little is known of these 
enzymes due to the difficulty in extracting the quantity of 
material necessary for enzyme purification, and to the 
difficulty in removing the other contaminating RNA polymerases 



13 



Most studies of organelle transcription therefore utilized 
in vivo labeling and experimentation. 

Structure 

RNA polymerase II has been purified from calf thymus, 
rat liver, sea urchin, yeast, mouse embryo cells, Hela 
cells, xenopus and maize (1,5,27). The subunit structure 
of the type II RNA polymerase from calf thymus was evalu- 
ated on SDS-polyacrylamide gel electrophoresis (38) . These 
subunits had molecular weights of 215,000, 185,000 and 
150,000 and were designated Bl, B2, and B3 . Gel densitom- 
etry indicated a ratio of 1:1:2 between the gel bands 
Bl:B2:B3 (39). These subunits apparently came from two 
subsets of RNA polymerase II. The first subtype gave 
bands Bl and B3, the second subtype gave bands B2 and B3. 
There were also 3 smaller subunits of molecular weights 
20,000, 30,000 and 40,000. The large subunits Bl, B2 and 
B3 appeared to be similar to the and 3 ' subunits of the 
E. coli RNA polymerase. It has been suggested that or-amanitin 
inhibits the type II RNA polymerase by binding to subunit 
B3. 

Ma i z e RNA Po lymer a s e 

Maize RNA polymerase was one of the first eukaryotic 
RNA polymerases solubilized (27) . Other eukaryotic RNA 
polymerases were solubilized from animal tissues (1) ; 
however, other plant RNA polymerases were studied primarily 



14 



.using plant chromatin or crude salt fractionated supernatants 
(40,41,42). RNA polymerase activity was first detected in 
the soluble fraction of a French pressure cell extract in 
1964 by Mans and Novelli (43) . The RNA polymerase in 
this extract was further purified by DEAE-cellulose chromo- 
tography using a linear Tris-HCl gradient (Stout and Mans 
1967) (27). The average specific activity eluted was 4.06 
nmoles AMP/mg at 10 rain. . This eluted RNA polymerase would 
utilize either native or denatured DNA as a template 
equally well, although at low DNA levels (10 ug DNA/ml) dena- 
tured DNA was eight times as effective as native DNA (44) . 
Denatured calf thymus DNA was more efficient as a template 
than denatured maize DNA; however, the calf thymus DNA had 
a much greater hyperchromicity than maize DNA, 17 percent 
vs 8.3 percent (44). The RNA polymerase required all four 

nucleoside triphosphates, a bivalent metal ion, and DNA to 

14 

incorporate [8- C]ATP into acid-insoluble material (27) . 
The metal could be either magnesium (25 mM) or manganese 
(5mM) . If [a-32p]uTP or [a-32p]ATP were used as labeled 
substrate, the nearest neighbor frequency indicated RNA 
containing all four nucleosidemonophosphates had been syn- 
thesized (27) . The reaction was inhibited by actinomycin D, 
pyrophosphate and DNase (43) . The product synthesized on 
native DNA was greater than 90 percent digested by pancreatic 
ribonuclease, while on denatured DNA this decreased to 73 
percent (27) . On sucrose density gradients the products 



•were 14-16 S, which corresponded to the distribution of 
the denatured DNA template (44) . This distribution was 
that expected for DNA-RNA hybrids. As expected, upon 
heating the complexes disaggrated and all nucleic acids 
were at the top of the gradient (4-6 S) . 

The RNA polymerase purified by the method of Stout 
and Mans was identified as a type II nucleoplasmic RNA 
polymerase by its sensitivity to a-amanitin (45) . This 
type II RNA polymerase did not synthesize homopolym.ers 
such as poly (A) with denatured DNA, as many bacterial 
RNA polymerases did (46), for with denatured DNA "the 
formation of a homopolymer was not detected with the maize 
polymerase'" (44, p. 752), nor was it detected with any 
other eukaryotic RNA polymerase. 

A type I maize RNA polymerase was reported by Strain 
et al . (47) . Maize leaves were used as crude material and 
carried through DEAE-cellulose chromotography . The type I 
RNA polymerase eluted at 0.08 M (NH^)2S0^ (47). Strain, 
et al_. also detected two overlapping peaks of activity in' 
the type II RNA polymerase region. ■ One peak preferred 
native . DNA, the other preferred denatured DNA as measured 
by total AMP incorporated (47) . The metal requirements of 
the leaf RMA polymerase indicated a preference for magnesium 
over manganese, with optimums at 25 mM for magnesium and 
8 mM for manganese (47) . 



Polyadenylic Acid 

Enzymatic Synthesis 

A eukaryotic polyadenylic acid polymerase was first 
discovered in calf thymus by Edmonds and Abrams in 1962 

(48) . Its activity was inhibited by the other nucleotide 
triphosphates, it required magnesium and it was particulate, 
perhaps bound to the nuclear membrane. Its product was 
almost pure poly (A) , except for about 1 percent of the 
adenylate residues which were joined to cytidylate resi- 
dues. The enzyme could not be freed of endogenous RNA. 
Others have purified poly (A) polymerases from rat liver 

(49) and from maize (50). The maize poly(A) polymerase 
adds poly (A) chains to the 3' hydroxyl of primer nucleic 
acids, either RNA or DNA (51) . 

Poly (A) synthesis has also been studied in prokaryotic 
systems. E. coli RNA polymerase, with denatured calf thymus 
DNA and only ATP as substrate, will synthesize poly (A) 
sequences using poly(dT) regions of the DNA as template 
(46). Reiteritive transcription of poly(dT) regions, each 
greater than 5 nucleotidyl residues long, resulted in the 
synthesis of long poly (A) chains through a repeated 
utilization of the poly(dT) template by an unknown mechanism 
Poly (A) synthesis required denatured DNA and was inhibited 
by the addition of the other nucleoside triphosphates. 
The polymerase would not lengthen added poly (A) primers (46) 



.The E. coli RNA polymerase had a greater affinity for 
denatured DNA than for native DNA (52) , perhaps reflecting 
a greater binding affinity for the exposed poly(dT) regions 
of the denatured DNA. 

Importance 

From 1961 to 1968, poly (A) synthesis by prokaryotic 
RNA polymerase was an unusual artifact of the assay and of 
unknown significance. With the discovery of poly (A) se- 
quences in ENA isolated from numerous eukaryotes; vaccinia 
virus cores (53), Hela cells (54,55), mouse sarcoma cells 
(56), and avian myeloblastosis virus (57), poly (A) syn- 
thesis again became of interest. In eukaryotes only a 
portion of the DNA- like nuclear RNA is transported to the 
cytoplasmic polysomes. In addition, the nuclear RNA is 
much larger in size than that found in the cytoplasm (53) . 
In studying poly (A) synthesis in vaccinia viral cores, 
Kates asked, "Could poly (A) sequences in nuclear RNA . 
play a role in either the cleavage of RNA into smaller 
pieces or in the selective transport of certain species 
to the cytoplasm?" (53, p. 752) . To answer the question 
of the role of poly (A) , two additional questions should 
first be answered. Is the poly (A) covalently attached to 
the RNA, and if so, where? If attached to RNA, what 
enzyme catalyzed the synthesis of the poly (A) sequence? 
Finding some poly (A) attached to RNA does not imply that 



-all the. poly (A) was attached, or remained attached. 
Knowing the time and location of poly (A) synthesis and the 
enzymes responsible would contribute significantly to an 
understanding of the physiological function of poly (A) 
synthesis. 

Location in Vivo 

Poly (A) can be isolated from the HnRNA or from the 
rapidly labeled RNA isolated from polyribosomes (54,55,56). 
Edmonds (54) indicated the data was consistent with the 
idea that every HnRNA contained at least one poly (A) se- 
quence » More than one mode of poly (A) synthesis was 
implicated since cordycepin (3-deoxyadenosine) suppressed 
the labeling of mRNA found on Hela ribosomes while not 
effecting the labeling of nuclear RNA (58). This suggested 
there may be two enzymatic activities that synthesized 
poly (A) ^ one sensitive to cordycepin that synthesized 
poly (A) and one insensitive to cordycepin that synthesized 
HnRNA containing poly (A) sequences . Lim and Cannelakis ' 
(59) results with haemoglobin mRNA indicated that, at most, 
it could contain 70 polypurine residues of 70 percent AMP. 
Furthermore, this polypurine sequence was not at the 3' 
end of the haemoglobin mRNA, since the 3' end contained 
only 7 or 8 AMP residues before the first pyrimidine (60) . 
Therefore, the poly (A) must be at the 5' end or inside the 
RNA chain, if there at all. 



Tiie strongest evidence for poly (A) being contained 
in newl2S synthesized RNA was that of Kates (53) . Utilizing 

' vaccixira''" cores inciibated in vitro, under conditions of RNA 
synthesis, Kates' data indicated that after mild RNase treat- 
ment all of the RNase resistant poly (A) sequences sediment- 
ated at 4 S and had a chain length greater than 5 nucleo- 
tides. The association of poly (A) with RNA was not dis- 
rupted by heating at 100°, nor by 75 percent DMSO at 80° 

-An the presence of cold poly (A). This indicated that, if 
in fact, the poly (A) was attached to the RNA it v/as probably 
through a covalent bond. When poly (A) was synthesized by 
vaccinia cores with only ATP as the substrate, then the 
poly (A) was not later attached to newly synthesized viral 
RNA (53). This in vitro poly (A) synthesis continued for 
only 5 min., the poly (A) was 18 nucleotides long and had ■ 
a uniform 5.8 S value. After alkaline hydrolysis and 
chromotography there was one 5 ' tetraphosphate and one 
adenosine for every 18 AMP residues (53) . If synthesis 
'tDCcurred in the presence of all 4 nucleoside triphosphates, 
25-3 percent of the AMP incorporated was in poly (A) . The 
poly (A) was synthesized without a lag period upon addition 
of the substrates, but if UMP incorporation was measured, 
there was a 1.5 min. lag before incorporation began (53). 
This indicated poly (A) synthesis preceded RNA synthesis. 
If the poly (A) synthesized in the presence of all 4 nucleo- 
side triphosphates was purified and alkaline hydrolyzed. 



there were 5 adenosine residues for every adenosine 
tetraphosphate (53). Therefore, if poly (A) was the initial 
sequence and contained the tetraphosphate, there were four 
more sequences that were synthesized internally or at the 3' 
terminis. 

Based upon hybridization of poly (A) to denatured 
vaccinia DNA, Kates estimated there could be greater than 
25 poly(dT) sequences in the vaccinia DNA, each 180 
nucleotides long (53) . This wag consistent with Heaust 
and Botchan (61) , who stated that 10 percent of the genome 
of mice consisted of AT-rich regions. The function of this 
DNA was not clear, but it was probably not transcribed in 
vivo (61). It was, however, known to be uniformly distri- 
buted among all chromosomes. 

Summary 

Poly (A) sequences are known to occur in vivo and in 
vitro . They appear to be synthesized by two enzyme systems, 
poly (A) polymerases sensitive to cordycepin, and by RNA 
polymerases insensitive to cordycepin. Some poly (A) se- 
quences are located at the 3' hydroxyl end of RNA polymers; 
however, there is evidence for both poly (A) sequences in- 
ternally and at the 5' end of the RNA. The importance and 
functional significance of these sequences is not firmly 
established. 



METHODS AND MATERIALS 



Methods 

RNA Polymerase Purification 

The purification of maize KNA polymerase has evolved 
through enumerable modifications of that published initially 
(27) . Each step in the procedure utilized here is indicated 
in Figure 1 . 

Step 1» Preparation of mater'ial 

Grain (4-10 liters) , in a plastic garbage can with a 
perforated bottom, was imbibed and germinated under run- 
ning water (23°) for five days xintil the shoots were 2 to 
4 cm long. The roots and shoots were separated from their 
kernels with a vibrating, stainless steel gravel separator 
under a shower, collected batchwise in a strainer, excess 
water was shaken out, and then the material was dropped into 
liquid nitrogen. Packets of frozen shoots and roots (90 g 
each) were wrapped in aluminum foil and stored in a Revco 
freezer (-76°) . - 

Step 2. Hom.ogenization 

One packet of seedling tissue was homogenized in a 
Waring blender in 135 ml of Buffer H for 60 sec. at low 

21 



22 




STEP I SEPARATE 




E 



ROOTS AMD SHOOTS! 



STEP 2 flOr-IOGEMIZE 



FILTER BOUUD 




STEP 3 CEriTRlFUGE 




SUPERMATAMT 



.STEP 4 50% CriH^}2S04 



PELLET 




□ 



SEPHADEX G50 
3_ 



EXCLUDED VOLUr^lE 



DEAE-CELLULOSE 



FLOW THROUGH 



RMA POLYyERASE 



Figure 1. PURIFICATION PROCEDURE FOR RNA POLYMERASE 



speed and for 15 sec. at high speed. The homogenized 
material had the consistency of a thick milkshake. The 
homogenized material was immediately filtered through four 
layers of cheesecloth, through a layer of miracloth and 
into a chilled Erlenmeyer flask. Material retained in the 
filters was discarded. 

Step 3. High speed centrif ugation 

The filtered homogenate containing 20 to 4 percent 
of the protein present in the shoots and roots was centri- 
fuged for 60 min. in a Ti50 rotor at 200,000 x g at 0°. 
The supernatant fraction was decanted into a chilled 
graduated cylinder through a layer of miracloth, which re- 
tained the lipid layer accumulated at the top of the centri- 
fuge tube. The supernatant fraction contained approximately 
50 percent of the protein present in the filtered homogenate 

Step 4. Ammonium sulfate precipitation 

To the high speed supernatant an equal volume of 
saturated (NH_^)2S0^ was added slowly with continuous and 
gentle stirring in an ice- jacketed beaker. After 30 min. 
of stirring the resulting precipitate was collected by 
centrif ugation (10,000 x g, 10 min., 0°) and resuspended 
in a minimal volume of Buffer R (approximately 15 ml) . 



24 

Step 5. Salt equilibration on sephadex 

...JEquilibration in Buffer E v/as accomplished by passage 
-trf- "the "'re suspended precipitate through a sephadex G50 
colman ■ ( 2 . 5 x 11 cm) equilibrated with Buffer E. The ex- 
cluded material was diluted to 25 ml with Buffer E for 
absorption to DEAE-cellulose . 

Step 6 . DEAE-cellulose chromotography 
-J-' The excluded volume eluted from sephadex G50 
chromotography was loaded (1 ml/rain.) onto a DEAE-cellulose 

(see materials) column (2.5 i.d. x 11- cm) equilibrated with 
Buffer E. The loaded column was washed with 6 ml of 
Buffer E and the flow rate vjas decreased to 0.25 ml/min. 
A 60 ml (NH^)2S0^ gradient (0.20 to 1.0 M) was begun 
immediately after passage of the 60 ml of Buffer E. 
Column eluates were monitored and recorded by a continuous 
flow ultraviolet-monitoring system (Gilford spectrophoto- 
meter and a Honeywell recorder. Figure 2) . Eluted fractions 
were collected directly from the flowcell into glass vials 

(2.5 ml/vial) and frozen in liquid nitrogen. 

Polymerase Assay 

RNA polymerase was assayed in a 0.10 ml standard 
reaction mixture containing: 10 ymoles Tris-Hcl , pH 7.6 @ 
25°; 0.25 ymoles each UTP , CTP , GTP, (sodium salts); 
0.10 ymoles [8--'-'^C] ATP (specific activity 1.7 to 4.5 yC/ymole) 
1.0 ymole MgCl^; 1.0 ymole 2-mercaptoethanol ; 8 ymoles 



25 




i 




1 

s 


O J 







i 



SPECTRO 
PHOTOMETER 





8^ 




GRADUATED CYL. 



CO 



< 
IT 
O 



-J 

o 

CO 



CD 
■H 



■ ' .. 26 

{NH^)2S0^; 0.37 ^260 ^^'^^ thymus DNA; 5 yg BSA and enzyme 
as indicated (27) . An assay mixture was prepared immediately 
before use and held on ice until the reaction was initiated 
by the addition of enzyme to the mixture. The reaction 
tube was immersed in a 30° water bath for 2 min. and acid- 
insoluble radioactivity was determined by a modification of 
a procedure previously described (27) . The reaction was 
terminated by pipetting the mixture onto a 3 MM filter 
paper disk. The reaction mixture was absorbed into the 
disk for 10 sec. under a heat lamp and precipitated by 
immersion in cold 10 percent TCA containing 2 mM sodium 
pyrophosphate. As many as 4 disks were then extracted 
five times with 100 ml of the TCA solution (5 min. each 
time), once with 100 ml ethanol-ether (1:1), and once with 
100 ml ether for 3 to 5 min. All extractions were per- 
formed at room temperature. The disks were then dried 
and counted in a scintillation solution containing PPO, 
POPOP and reagent grade toluene. Disks which received 
reaction mixture containing no enzyme averaged 15 cpm 
above machine background. The wash procedure reduced the 
cpm from approximately 400,0 00 cpm/disk to 35 cpm/disk 
for the no enzyme control. Machine counting efficiency 
was 0.75 cpm/dpm. One unit of RNA polymerase activity is 
defined as 1 nmole AMP incorporated into acid-insoluble 
material under standard reaction mixture conditions in 
20 rain. 



■;\ 27 

Protein Determination 

Protein was measured on samples precipitated with 
10 percent trichloroacetic acid by the method of Lowry 
et al . (62) , using BSA as a standard. 

Ribonuclease Treatment 

Pancreatic ribonuclease (80 yg/ml) , heated at 8 0° 
for 10 min. to inactivate DNase, was added to 0.1 ml aliquot 
of an incubated reaction mixture to a final concentration 
of 20 pg/ml, and incubated for an additional 10 min. at 
30° (54) . Acid-insoluble material remaining was determined 
on filter paper disks with the polymerase assay wash 
procedure (see polymerase assay) . 

Alkaline Hydrolysis 

Product, isolated by sephadex gel filtration or by 
KCIO^ precipitation from an incubated reaction mixture, 
was suspended in 1.0 ml of 0.3 M KOH and incubated at 37° 
for 18 h in a stoppered test tube. After the 18 h hydrol^^^i 
the solution was acidified with 7 percent HCIO^ and the 
precipitate removed by centrif ugation. The supernatant 
was neutralized with 0.3 M KOH and the salt was removed 
from the hydro lysate by charcoal adsorption (63) . The 
charcoal column was washed successively with water ( 3 ml) 
and 0.1 M NH^OH (5 ml) and the nucleotides eluted with 
5 ml of ethanol-NH^OH (ethanol-conc NH^0H-H20, 2:1:2 by 
volume) . The eluted fraction was lypholyzed to dryness and 
resuspended in water. 



• . 28 

Paper Electrophoresis 

Samples to be electrophoresised were adsorbed to 
paper strips (4 x 30 cm, Whatman #1) by repeatedly apply- 
ing and drying on one spot (5 to 10 mm dia) . The paper 
strips were then subjected to electrophoresis in 0.025 M 
sodium citrate, pH 3.5, for 4 h at 300 v, 6-8 amps, at 
25° in a Universal Electrophoresis. Cell (Buchler Instru- 
ments) . 

Polyacrylamide Gel Electrophoresis 

Polyacrylamide gels for native proteins were prepared 
and run according to the method of Davis (64) using a 
stacking gel and 5.2 percent polyacrylamide gels. The 
gels were run at pH 8.8 and stained with aniline blue 
black. The 10 percent polyacrylamide gels and denatured 
enzyme protein were prepared and run according to the 
method of Weber and Osborn (65) . The 10 percent gels were 
run at pH 7.0 in 0.1 percent SDS and stained with coomassie 
brilliant blue. 

Materials 

DEAE-cellulose, Bio-RA.D, Cellex-D (6.1 meq per g, 
dry weight) , was washed by the method of Peterson and Sober 
(66), equilibrated in 0.05 M Tris-HCl, pH 7.6 at 25°, and 
stored at 4°. Sephadex G50, medium grade, Pharmacia, was 



swoll^ii..,.2ria .eg.uil-lbrated in 0.05 M Tris-HCl, pH 7.6 at 25°, 
aE.d'^ox0A>-ak{p^-:^,i^movine serum albximin, 5X crystallized, 
was.:.;£rom PenteK*^BlGchemicals . The nucleoside triphosphates 
GTP, CTP, UTP 'arid ATP were purchased as sodium salts from 
Schwarz/Mann, including [S-l^cjATP, [2-l'^C]UTP and [a-32p] 
ATP. Calf thymus DNA was purchased from Schwartz/Mann, and 
pancreatic ribonuclease , chromatographically pure, was 
purchased from Worthington Biochemicals . Actinomycin D 
was -purchased,;-f'xsan Schwarz/Mann and cordycepin, grade C, 
was purchased from Sigma. Alpha-amanitin was a gift of 
Dr. T. Weiland and rifampicin was a gift from Gruppo- 
Lepetit S .P . A. JResearch Laboratory. Zea mays L., WF9 x 
Bear 38, v/axy, was purchased from the Bear Hybrid Seed Co. 

Reagents 

Calf thymus DNA was dissolved in 0.1 x SSC at 37 ^26(y 
ml and stored in 0.2 ml aliquots at -17° (67). An aliquot 
was thawed for use before each assay. Denatured DNA was 
prepared by the dilution of a freshly thawed aliquot of 
DNA in 0.1 x SSC (1:1 v/v) into a sealed vial, 5 min. 
immersion in boiling water, followed by quick chilling in 
iced-water (average hyperchromicity 24%) . All the ^260 
measurements in the tables and figures represent the 
absorbancy before denaturation. 

Stock solutions of 1,0 M Tris-HCl, pH 8.0 or pH 7.6 
at 25°, 1.0 M MgCl2/ 14.7 M 2-mercaptoethanol were used to 
make the foil-owing buffers: Buffer H, 0.25 M sucrose. 



30 



0.10 M Tris-HGl, pH 8.0, 1.0 mM MgCl2, and 50 mM 2-mercap- 
toethanol; Buffer E, 0.05 M Tris-HCl , pH 7.6, 10 mM 
2-mercaptoethanol and 0.20 M (NH^)2S0^; Buffer R, 0.05 M 
Tris-ECl, pH 7.6, 10 mM 2~mercaptoethanol . Buffers were 



made immediately before use, using warm glass~distilled 
water to m.inimize gas bubbles in the column eluates passing 



Saturated ammonium sulfate was kept at 4° with 
crystals visible in the bottom of the bottle. Before use 
in protein precipitations, concentrated NH^OH was added 
drop-wise until a 1:20 dilution of the (NH^)2S0^ was pH 8 
at 25°' (67) . Sodium chloride-sodium citrate buffer (SSC) 
1 X, was 0.15 M NaCl, 0.015 M sodium citrate, pH 7.3 at 
25° (68) . 



through the spectrophotometer flowcell. 




RESULTS 



- RNA Polymerase Purification 

Utilizing the RNA polymerase .purification procedure 
described in METHODS, RNA polymerase was purified 200-fold 
over the 200,000 x g supernatant activity, or 1,000-fold 
over that present in the tissue homogenate. The results 
of several RNA polymerase purifications are summarized in 
Table 1.' The steps in purification correspond to those 
in Figure 1. In 10 h, 2 mg of RNA polymerase were purified 
frcm a 90 g packet of maize roots and shoots, with a re- 
coveary from 55 percent to 69 percent of the initial AMP 
incorporating activity present in the 200,000 x g super- 
natant. Variability in protein and activity of the 200,000 
X g supernatant probably resulted from variability in 
horaogenization v/ith the Waring blender. Activity detected 
in the 50 percent (NH^)2S0^ precipitate was variable . 
(Table 1) , resulting from the presence of nucleases and 
variable amounts of salt in the resuspended protein. After 
salt equilibration on sephadex G50, assays of polymerase 
activity were less variable. 

Both gradient and batch elution of DEAE-cellulose 
resulted in highly active RNA polymerase. Gradient- eluted 
RNA polymerase had a higher peak specific activity (104 nmoles 



32 



TABLE 1 

STEPS IN UNA POLYMERASE PURIFICATION 



A standard reaction mixture contained 0.37 ^260 ■^•'^ umole 

MgCl2, and enzyme__as follows: 96 yg 200,000 x g supernatant, 
500 yg 50% ASP, 250 yg sephadex G50, or 3 to 5 yg DEAE- 
cellulose fraction. 



Step 



. . Specific Total v-i^i^ 
Protein , ■ ■ j_ j.- ■ i. Yield 

Activity Activity 



(mg) (nmoles AMP/mg) (units) {%) 



200,000 X g Supernatant 
.a 



50% ASP 



a 



Sephadex GSO 
DEAE-cellulose 

Gradient elution"^ 
Batch elution 



507+100 0.57±0.12 290±90 
207± 50 0.72+0.3 195±60 
250± 50 0.70±0.07 175±20 



2.0 
5.0 



66.0 
40.0 



132 
200 



100 
67±30 
60±10 

55 
69 



Average of 3 preparations. 

^Flowthrough contained less than 5% of added RNA polymerase 
activity. 

"^Gradient elution, peak tube specific activity, 10:4 timoles 
AMP/mg; protein, 0.2 mg/ml . 

"^Batch elution (0.4 M (NH^)2S0^), peak tube . specif ic activity, 
90 nmoles AMP/mg protein 0.5 m.g/ml . 



I 

33 



AMP/mg)', however, it was more dilute (0.2 mg/ml) and its 
preparation more time consuming (6 h) . Batch eluted RNA 
polymerase had a lower peak specific activity (9 nmoles AMP/ 
mg) , but it was more concentrated (0.5 mg/ml), more rapidly 
eluted (2 h) and the net recovery was better than with the 
gradient procedure. 

The profile of A2gQ material during gradient elution 
of the DEAE-cellulose column indicated most of the material 
failed to bind to the resin equilibrated with 0.2 M (NH^)2 
SO^ (Figure 3). However, all of the RNA polymerase activity 
was bound to the resin. Of the two partially resolved 
peaks of ^280 absorbing material eluted from the resin by 
the {NH^)2S0^ gradient, shown in more detail in Figure 4, 
only the second peak exhibited RNA polymerase activity. 
Although the RNA polymerase activity detected with dena- 
tured DNA was twice that detected with native DNA, the 
ratio of specific activities with native and denatured 
DNA was constant among the eluted fractions of the second 
peak. Attempts to enhance purification by equilibration 
of the resin with higher than 0.2 M (NH^)2S0^ to reduce 
binding of the protein in the first peak (Figure 4) were 
unsuccessful. If the salt concentration was increased to 
0.21 M, 15 percent of the enzyme activity appeared in the 
column flowthrough. 




VOLUf^E ml 



Figure 4. RNA POLYMERASE ACTIVITY IN 
DEAE-CELLULOSE FRACTIONS 

Assayed in a standard reaction mixture 
containing 0.5 ui^oles MnCl^, 0.037 ^260 
DNA; either denatured (solid triangles) 
or native (open triangles) and "^f to 4 yg 
enzyme. 



36 



Polyacrylamide Gel Electrophoresis 

, Aii-'-ialiquot of the proteins showing the highest RNA 
''p'olymera&e specific activity (eluted from DEAE-cellulose 
■•at 235 'ml in Figure 4) was examined by electrophoresis on 
5.2 percent polyacrylamide gel. Several components were 
visible, with more than 10 bands staining with aniline 
blue black (Figure 5) . All the protein staining material 
migrated into the gel , toward the anode at pH 8.8. There- 
fore, the "proteins are acidic and none were excluded by 
the 5.2 percent polyacrylamide gel. The slowest migrating 
and darkest staining band, however, may represent an 
gregate of some of the more rapidly moving components . The 
intensity of this major band was approximately equal to 
that of the BSA standard (20 ug) and, therefore, may account 
for 50 percent of the added protein. If an aliquot of the 
same protein preparation was denatured with 0.1 M 2-mercap- 
toethanol and 0.1 percent SDS, then subjected to electro- 
phoresis on 10 percent polyacrylamide-0 . 1 percent SDS gels, 
the number of visible bands was reduced to 5 (Figure 6) . 
Two distinct bands, migrating 13 and 14 mm into the gel, a 
faint band at 19 mm ^d a still fainter pair of thin bands 
at 21 and 2 3 mm were characteristic of all the purified 
maize RNA polymerase preparations. The decrease in detect- 
able bands on SDS-polyacrylamide as compared to the 5.2 per- 
cent polyacrylamide gels may reflect disaggregation of the 
denatured proteins. Alternatively, the presence of more . 



A W B 



Figure 5. POLYACRYLAMIDE GEL ELECTROPHORESIS OF 

NATIVE RNA POLYMERASE 

Samples were run on 5.2% polyacrylamide gels at pH 8.8 
(see METHODS) . Gel A contained 4 yg peak RNA poly- 
merase (eluted at 235 ml. Figure 4 ) . Gel B contained 
20 yg ESA. 



(+) 



Figure 6. POLYACRYLAMIDE GEL ELECTROPHORESIS OF 
DENATURED SNA POLYMERASE 

Samples were denatured in 0.1% SDS, 0.14 M 2-mercapto 
ethanol at 37° and run on 10% polyacrylamide gels 
containing 0.1% SDS at pH 7.0 (see METHODS). Gel A 
contained 20 ug peak RNA polymerase (eluted at 235 ml 
Figure 4). Gel B contained 6.2 yg denatured BSA. 
Gel C contained denaturing buffer only. 



■ 39 

bands on the gel run with native protein may reflect greater 
sens±±.i';yi:ty of band detection since the sample analyzed con- 
■■ira'ine'd'''6:wice as much protein and the gel was stained with 
aniline blue black rather than coomassie brilliant blue. 
Assuming that the distance migrated by the denatured protein 
components in SDS-polyacrylamide gels was a function of 
their molecular weights, then the pair of bands migrating 
21 and 23 mm (equivalent to the migration of BSA) were 
about 65,000 to 75,000 d. Therefore, the other bands 
characteristic of the RNA polymerase preparations corre- 
sponded to polypeptides of higher molecular weight. 

Enzyme Stability 

Storage temperature and freeze-thaw 

Maize RNA polymerase collected in glass vials and 
immediately stored in liquid nitrogen lost no activity 
after 3 months. The enzyme was stable for 19 days at -76°, 
butibst all activity at -17° (Table 2, part A). The 
addition of glycerol to 20 percent by volume to fresh 
enzyme preparations did not stabilize the polymerase 
enough to make storage at -17° practical. However, in 2 
percent glycerol the enzyme did retain 5 percent activity 
for 17 h at 4° (Table 2, part B) . The enzyme stored in 
liquid nitrogen was stable to repeated freeze-thaw. The 
loss of activity observed may reflect decay accumulated 
during the time the enzyme was held on ice preceding assay 
(Table 2, part C) . 



40 



TABLE 2 

STORAGE AND FREEZE-THAW LABILITY OF RNA POLYMERASE 



After the indicated treatment aliguots of one enzyme 
preparation were assayed for RNA polymerase activity in a 
standard reaction mixture containing 1.0 ymole MgCl,., 



0.37 ^260 ^^'^^^^ ^NA and 18 yg enzyme. 100% incorporation' 
was equivalent to 172 pmoles AMP incorporated per reaction. 

% Incorporated 

A) Temperature (19 days) 

-76° (Revco freezer) 98 

-17° (Freezer) .1 

B) Glycerol Storage^ (17 h) 

-196° (Liquid nitrogen) " 96 

-17° (Freezer) 78 

4° (Refrigerator) 48 

b 

C) Freeze- thaw 

One 100 

Two 100 

Three 86 

Four 82 

Five "^^ . 

storage on Ice (5 h) ' ' 80 



^Glycerol was added to 20% by volume to the enzyme preparation 
just prior to storage. 

A vial of enzyme frozen in liquid nitrogen was thawed at 37° 
and held on ice prior to assay. After assay it was refrozen 
in liquid nitrogen. This sequence was repeated as indicated 

^Enzyme frozen and thawed once prior to 5 h storage on ice. 



41 

Salt pre:clp:itation , . ■ 

,„ -...-f In:v-s"»va^?i"e^^^^^ to concentrate the RNA polymerase eluted 
<^roia' DEL£E-:cel^m.lose, solid (NH^)2S0^ was added to 8 per- 
cent saturation to the pooled fractions. Only 10 to 15 per- 
cent of the eluted activity was recovered in the precipitated 
protein. To facilitate precipitation, calf thymus DNA was 
added in varying amounts to the pooled fractions just before 
salt addition. Addition of DNA resulted in an increase in 
precipitated activity, roughly porportional to the amount 
of DNA added (Table 3) . Low recovery of polymerase in 
precipitates from a solution with high DNA levels (300 yg/ 
ml) suggested ...formation of a DNA-prote in complex soluble in 
high salt. Neither polymerase activity in the supernatants 
nor protein in the precipitates was determined, therefore 
inactivation of polymerase and specific activity were not 
assayed per se. 

Summary 

DNA-dependent RNA polymerase was purified 1,000-fold 
from an homogenate of maize shoots and roots. Following 
DEAE-cellulose chromotography the enzyme specific activity 
was greater than 10 nmoles AMP incorporated/mg enzyme at 
2 min. The recovery was greater than 5 percent of the 
activity detected in the high speed supernatant. Poly- 
acrylamide gel electrophoresis of both native and SDS- 
denatured proteins indicated the RNA pol^erase preparation 
still contained several polypeptides. Nevertheless the 



TABLE 3 



SALT PRECIPITATION OF RNA POLYMERASE 



Each sample contained 0.75 mg protein before 80% saturation 
with solid, powdered (NH^)2S0^. Just before addition of 
salt, DNA was added as indicated, then salt was slowly add- 
ed while the mixture was stirred on ice for 15 min. The 
precipitate was collected by centrifugation (10,000 x g for 
10 min. ) / resuspended and desalted in Buffer R on sephadex 
G25. Samples were assayed in a standard reaction mixture 
with 1.0 vtmole MgCl2 and 0.37 A26O 



DNA Added (yg/ml) % Activity Recovered^ 



None 



13 



2.4 



20 



'6.0 



30 



12.0 



38 



30.0 



46 



300. 



b 



10 



'Control incorporated 18 5 pmoles AMP. 
Very little precipitate obtained. 



43 



denatured protein exhibited those bands characteristic of 
eukaryotic RJES. "polymerases . The enzyme wa:s stable after 
"freezing ano*' -storage at -76°. Precipitation of the purified 
enzyme with"(NH_^) 2S0^ was facilitated by DNA. Further 
resolution of the proteins in the RNA polymerase preparation 
was thwarted by the instability of the enzyme at 4° and by 
the lack of recovery of active enzyme following salt 
precipitation. 

RHA Synthesis 

Assay Requirements 

Enzyme purified by the procedure described in METHODS 

exhibited the expected requirements for RNA synthesis. 

DNA, metal and all four ribonucleosidetr iphosphates were 

required for incorporation of labeled AMP into acid-insoluble 

material (Table 4) . Denatured DNA was utilized as well as 

native calf thymus DNA^, and maize DNA (not shown here) sup- 

2+ 

ported comparable activity. Mn satisfied the metal re- 

2+ 2+ 
quirement as well as Mg , in fact, better than Mg when 

denatured DNA was provided as the nucleic acid component. 

Incorporation of AMP in the absence of NTPs was significant, 

especially when assayed in the presence of denatured DNA. 

All of the radioactivity detected in acid-insoluble material 

on filter paper disks was dependent upon the addition of 

enzyme protein. ' , ' 



44 



TABLE 4 

ASSAY REQUIREMENTS FOR MAIZE RNA POLYMERASE 



A Standard reaction mixture contained 0.37 ^260 ^'^ ymole 

MgCl- or 0.5 lamoles MnCl^ and 5 ug RNA polymerase. 



System 



AMP Incorporated (pmoles) 
Native DNA Denatured DNA 



CoiE^lete, 

Cois^lete less DNA 

2+ 

Corr^lete less Mg 

2+ 2+ 
CoiH^lete less Mg , plus Mn 

ConK>lete less NTPs 

Complete less enzyme 



295 



300 
10 
. 



327 
1 
1 

551 
58 




^Total radioactive AMP incorporated per reaction mixture. 



DNA ... , 

■•■^^'SCEn- >-Mie^-:ctMp±ete system with Mg as the metal cof actor, 
■the"^®L' requirement- was satisfied with either native or 
heat-denatured' DNA (Table 4). The reaction was saturated 
with denatured DNA in excess of 0.4 A2gQ/ml, whereas with 
native DNA more than a 5-fold higher concentration (1.8 ■^2 6(/ 
ml) was required to reach the same level of activity (Fig- 
ure 7) . If equal amounts of native and denatured DNA were 
combiu'ed. and .'iassayed in the same reaction mixture, the 
activity measured could be accounted for by the denatured 
DNA alone. Thus, the denatured DNA appeared to out-compete 
±he native DNA: for the RNA polymerase. However, at 
saturating concentrations native and denatured DNA supported 
equivalent AMP incorporation. 

If MnCl2 was substituted for MgCl2 in the reaction 
mixture, there was almost a 2-fold increase in RNA polymerase 

activity with denatured DNA (Table 4) . In the presence of 
2+ 

Mn , the activity with denatured DNA was twice that with 
native DNA over a range of DNA concentrations (Figure 8). 
Although the RNA polymerase assay was saturated at 0.4A2gQ/ 
ml with both native and denatured DNA, AMP incorporation 
after 20 min. was not equivalent. Therefore, the concen- 
tration dependence of the polymerase on native and denatured 

2+ 

DNA dif fer-ed when assayed with Mg , but the maximum level 

2+ 

of 'acti-vi±y' was the same. In contrast with Mn the con- 
centration dependence with native and denatured DNA was 




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■ ..' , . . ■ 48 

similar but the maximum level of AMP incorporation differed. 

Metal 

For maize RNA polymerase activity there was an 

absolute 'requirement for a metal ion cofactor satisfied 
2+ 2 + 

With either Mg or Mn (Table 4) . There was a broad 
2+ 

Mg optimum concentration from 10 'to 14 mM, centered at 

12 ieM, and independent of the nativity of the DNA (Figure 9) . 

Routinely, however, 10 to 25 percent more activity was 

observed with denatured DNA than with native DNA. The 

2+ 

maxxmuiu AMP incorporation occurred with 5 mM Mn , either 

with native or denatured DNA (Figure 10) . With denatured 

DNAy the RNA polymerase activity was twice that with native 
2+ 

DNA at all Mn levels. As is characteristic of polymerases, 
the Mn optimum concentration was sharper than that of 
Mg . . 

Substrate 

With native DNA, 9 5 percent of the AMP incorporation 
required the presence of the NTPs (Table 4) . The RNA poly- 
merase reaction was satur£^ted with the pooled NTPs above 
1.0 mM for each of the three nucleoside triphosphates; 
GTP, CTP and UTP and no nucleotide inhibition was detected 
with a 5-fold excess (Figure 11) . The standard reaction 
was 2.5 mM with each NTP ; thus the assay of polymerase was 
not limited with respect to unlabeled nucleosidetriphosphates 

The RMA polymerase reaction mixture was saturated 




MAGr4ESlUi\1 CHLORIDE mU 



Figure 9. MAGNESIUM TITRATION WITH 
NATIVE AND DENATURED DNA 

Assayed in a standard reaction mixture 
containing 0.37 ^260 native (solid 

circles) or denatured (solid triangles) , 
6.4 vg RNA polymerase and MgCl2 as 
indicated. 



SOOf 




MANGANESE CHLORIDE mM 



Figure 10. MANGANESE TITRATION WITH 
NATIVE AND DENATURED DNA 

Assayed in a standard reaction mixture 
containing 0.37 ^260 native (solid 

circles) or denatured (solid triangles) , 
6.4 ug RNA polymerase and MnCl^ as indicated. 



51 




Figure 11. NTP TITRATION OF RNA POLYMERASE 

Assayed in a standard reaction mixture con- 
taining 1.0 iiiTioles MgCl2/ 0.37 ^260 ^^^^^^ 
DNA, 6.4 yg RNA polymerase and pooled NTPs 
(GTP, CTP, UTP) as indicated. 



52 



with ATP above 1 . mM (Figure 12). A Lineweaver-Burke 

-4 

reciprocal plot resulted in a of 1.25 x 10 M ATP 

(Figure 13) . The K was the same with native and dena- 

tuxed DNA, although the V with denatured DNA was twice 
' ^ max 

that with native DNA. The standard ATP concentration for 
the RNA polymerase reaction mixture was 1.0 mM, since it 
resulted in the highest ATP specific activity while main- 
taining the maximum rate of RNA synthesis. The rate of 
AMP incorporation decreased when the ATP concentration was 
below saturation. If the ATP concentration was decreased 
to 0.5 mM there was 86 percent of the maximum rate and if. 
it was decreased to 0.25 mM there was 68 percent of the 
maximum rate. The pmoles of AMP incorporated per reaction 
was independent of the [8-l'^C]ATP specific activity (see 
APPENDIX A. ) . ' 

Salt 

As with the partially purified enzyme, the more 
highly resolved preparations exhibited a dependency on 
added (NH^)2S0^ for RNA polymerase activity, especially 
in the presence of native DNA (Figure 14) . The maximum 
level of activity occurred between 60 and 9 mM (NH^)2S0^ 
with native DNA, and up to 100 mM with denatured DNA. 
Because the RNA polymerase fraction contained 0.4 M 
(NH^)2S0^, the titrations at concentrations below 4 mM 
were not performed. 




Figure 12. ATP TITRATION OF RNA POLYMERASE 

Assayed in a standard reaction mixture containing 
1.0 ymole MgCl2/ 0.37 A^^^ native DNA, 5 yg RNA 
polymerase and ATP as indicated. 




4 8 

■ ! . 
ATP 

Figure 13. LINEWEAVER-BUKKE PLOTS OF ATP 
■ . . TITRATIONS 

Assayed in a standard reaction mixture con- 
taining 0.5 umoles KnCl^r 0.37.A2gQ DNA, either 
native (solid circles) or denatured (solid 
triangles), and 4 yg RNA polymerase. The ATP 
concentration was mM with the K at 0.125 mM. 
The V was 100 moles AMP/mg/2U min. for de- 
naturei^DNA and 5 nmoles AMP/mg/2 min. for 
native DNA. 



55 




Figure 14. AMMONIUM SULFATE TITRATION OF 
RNA POLYMERASE 

Assayed in standard reaction mixture con- 
taining 1.0 ymole iMgCl2, 0.37 A^^q DNA, 
either native (solid circles) or denatured 

(solid triangles), 6.4 yg RNA polymerase and 

(NH,)^SO. as indicated. 



56 



. Enzyme 

If enzyme protein was not added there was no AMP 
incorporation in the standard RNA polymerase reaction 
mixture (Table 4) . The total AMP incorporated in 2 min. was 
the function of the enzyme protein added, up to 6 yg pro- 
tein per standard reaction mixture .(Figure 15). In the 
presence of the NTPs, the amount of AMP incorporated with 
denatured DNA was twice that incorporated with native DNA, 
at all enzyme concentrations. 

Rate of AMP Inco rp oration 

The amount of AMP incorporated in a standard RNA 
polymerase reaction increased as a function of time for 
more than 90 min. (Figure 16) . The rate of AMP incorpora- 
tion decreased continuously during the initial 2 min. of 
incubation and then became constant. . The initial rate 

(R-j^) and the late rate (R2) were arbitrarily defined as 
that occurring up to 20 min., and that occurring after 
20 min. What appeared to be two different reactions 

(initial and late) were not resolved by increasing the 
incubation temperature from 30° to 37°. 

Nearest Neighbor Frequency 

Product synthesized on denatured calf DNA with 
[a-32p]ATP after alkaline hydrolysis, yielded four labeled 
nucleotides (Table 5) . Since the product was acid-insoluble 
and excluded from sephadex G25, indicating an oligomer, 
the nearest neighbor frequency data indicated the product 



57 




PROTEIM pg 



Figure 15. RNA POLY^iERASE ACTIVITY AS A 
FUNCTION OF ENZYI4E CONCENTRATION 

Assayed in a standard reaction mixture con- 
taining 0.5 liinoles MnCl2, 0.37 A^gQ DNA, 
either native (solid circles) or denatured 
(solid triangles) , and enzyme protein as 
indicated. 



58 




50 100 

TIME mln 



Figure 16. RATE OF AMP INCORPORATION AS A 
FUNCTION OF TIME 

Assayed in a standard reaction mixture (7X) each 
containing 1.0 ymole MgCl2/ 0.37 ^2 60 ^^"^^"^^ D}>!A 
and 18 ug Rl-JA polymerase. The reactions were 
incubated at either 30° (solid circles) or 37° 
(solid triangles) and 0.1 ml aliquots removed at 
the times indicated. 



59 



TABLE 5 

NEAREST NEIGHBOR FREQUENCY ANALYSIS OF PRODUCT 
SYNTHESIZED WITH DENATURED DNA 



A standard reaction mixture containing 0.37 ^2S0 ^^j^^^^ured 
DNA, 0.10 ymoles [a-32p]ATP (specific activity 30 mCi/ininole) , 
0.4 ymoles MnCl2 and 10 ug enzyme was incubated at 30° for 
60 min. 1% SDS was added to the reaction, it was passed 
over sephadex G25, the material in the excluded volume was 
hydrolyzed in 0.3 M KOH for 17 h at 37°, adsorbed to acti- 
vated charcoal and the eluted nucleotides lyophilized, re- 
suspended in H2O and resolved by paper electrophoresis, 
(see IffiTHODS) . 



CpA ApA GpA UpA 

Added Recovered^ 



55,000 27,600 0.25 0.44 0.18 

^Some of the sample was lost during lyophilization. 



0.13 



was RNA. The relatively high ApA frequency derived from 
product synthesized with denatured DNA suggested the 
..presence of poly (A) regions in the product. 

Summary 

The maize RNA polymerase required added DNA, the 

four nucleosidetriphosphates; ATP, GTP, CTP and UTP, and 

2+ 2+ 

a metal, either Mg or Mn , for activity. The maximum 

■activities with native and denatured DNA were equal when 
2+ 

Mg was a cof actor. The maximum activity with denatured 

2+ 

DNA was twxce that with native DNA if Mn was the metal 
cof actor. Enzyme activity was salt-dependent and pro- 
portional to the amount of enzyme protein added. The 
substrate reached saturation above 1.0 mM and the reaction 
continued for at least 9 min. The product with denatured 
DNA had a high ApA nearest neighbor frequency. 

Polyadenylic Acid Synthesis 

The RNA synthesis data indicated that an additional 
activity might be present in the RNA polymerase reaction. 
These indications were: 1) With denatured DNA there was 
significant incorporation of AMP in the absence of the NTPs. 

2) There was a doubling in activity with denatured DNA 

2+ 2+ 
when Mn replaced Mg , but with native DNA there was littl 

change. 3) There appeared to be a biphasic rate of AMP 



61 

incorporation, one rate at early times and another at late. 
These a:EBi,,ri-t£ -.suggested poly (A) might be synthesized early 
in a ai-eaction . mixture containing denatured DNA. 

Assay Requirements 

The standard reaction mixture for poly (A) synthesis 
was the same as that for the RNA polymerase assay (see 
METHODS) -except the NTPs were omitted. 

DNA . - 

2+ 

With Mg as cof actor, AMP was incorporated with 

denatured DNA in the absence of the NTPs (Table 4) . With 

denatured DNA the reaction was saturated at 0.2 Aom/nil 

z 6 

(Figure 17) , or about half that required to saturate the 

reaction in the presence of NTPs (Figure 7) . The maximum 

AMP incorporated with native DNA did not exceed 5 percent 

of the AMP incorporated with denatured DNA. 
2+ 

With Mn as cof actor, there was a 2-fold increase 

in AMP incorporation with denatured DNA as compared with 
2+ 

..Mg , but the denatured DNA still saturated the reaction 
mixture at 0.2 A2gQ/ml (Figure 18). Native DNA supported 
a much lower rate of incorporation ranging from 8 percent 
to 15 percent of the activity with denatured DNA. 

Substrate 

AMP incorporation in the absence of the NTPs was 

first detected with denatured DNA (Table 4) . In the 

2+ 2+ 
presence of Mn , rather than Mg , only half of the AMP 



62 



CM 



t3 




a 



EH 

in 
O 

W 

u 

H 
CO 

CQ 
< 

Eh 
H 
D 

H 
03 
W 



Eh 

H 
12 

U3 

O 

H 
Eh 

EH 

H 
Eh 

< 

Q 



0) 
13 

In 



4) 

g «} 
>i o 

O 

P4 IS 



CD 

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0) 

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cn 

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0) 



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Q) ^ 
u cn 

-P H 

X o 

•H M 
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O 

c 

O 

-H -H 

O O 

rd m 
u 



> 

J-t -H 
(tJ -P 

c c 

+J iH 

m 0) 

fd -P 

■H 

C 0) 
-H 

■73 <; 

>iQ 
(C 

m 13 
tn C 
< (d 



63 






CM 1 


cn 


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ft 


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<! 




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tc 


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cn 


o iz; - 




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CU tfl 


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u c 


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H 




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fi nj !^ 






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[±4 





64 

incorporation on denatured DNA was dependent upon the 
other NTPs (Table 6) . In contrast essentially all the 
incorporation of radioactive UMP required the other NTPs. 
These results were consistent with the simultaneous syn- 
thesis of polyadenylic acid and RNA. Furthermore, they 
suggested that homopolymer synthesis might be limited to 
poly (A) . 

A titration of the NTP requirement indicated that 
at low NTP concentrations (0.025 mM) AMP incorporation 
was inhibited, whereas at higher NTP concentrations AMP 
incorporation was stimulated (Figure 19) . Except for the 
activity observed with no NTPs, the stimulatory portion 
of this titration resembled that with native DNA (Figure 11) 
Addition of GTP, CTP or UTP individually at 3 mM resulted • 
in 80 percent inhibition of AMP incorporation (Table 7) . 
The addition of UTP at 0.3 mM (12% of the standard assay 
concentration) inhibited AMP incorporation to the same 
degree. The inhibitory effect of low NTP concentrations 
was observed upon addition of the NTPs before or after 
addition of the enzyme and incubation of the reaction mix- 
ture (Table 8). If 0.025 mM NTPs (1% standard NTP con- 
centration) were added just after enzyme addition, after 
1 min. incubation or after 5 min. incubation at 30°, in 
all cases it resulted in inhibition of AMP incorporation 
(Table 8) . The total AMP incorporated per reaction 
increased the longer the addition of NTPs was delayed, 



65 



TABLE 6 

AMP Aira UMP INCORPORATION BY RNA POLYMERASE 



A standard reaction mixture containing 0.37 A^^q denatured 
DNA, 0.4 ymoles MnCl2/ either 0.10 umoles labeled ATP or 
0.05 ymoles labeled UTP and 4 yg enzyme. 



Labeled Nucleotide Incorporated' 

System (pmoles) 

14 14 

C-AMP C-UMP 



Complete 381 22 

b c 
Complete plus NTPs 913 458 



^Total radioactive nucleotide incorporated per reaction 
mixture . 

^NTPs contained 0.25 ymoles each: GTP^ CTP and UTP. 
^NTPs contained 0.25 ymoles each: GTP , CTP and ATP. 




Figure 19. NTP TITRATION WITH DENATURED ^ DNA 

Assayed in a standard reaction mixture containing 
0.037 A^,» denatured DNA, 0.5 umoles MnCl2 5 yg 
RNA polymerase and , pooled NTPs (GTP, CTP , and UTP) 
as indicated (mM each) . 



TABLE 7 



INHIBITION OF POLYADENYLIC ACID SYNTHESIS 
BY NUCLEOTIDES 



RNA polymerase activity measured in a standard reaction 
TP;ixt-ure containing 0.037 A^gQ denatured DNA, 0.05 ymoles 
■^MaCl-, 5 yg enzyme, and nucleotides as indicated. 



Additional Nucleotides AMP Incorporated^ (pmoles) 



GTP, CTP, UTP (0-. 25 ijmoles each) 619 

None 210 

UTP (0.3 ymoles) 40 

GTP (0.3 ymoles) 31 

CTP (0.3 ymoles) 43 

UTP (0.03 ymoles) SO 



^Total pmoles AMP incorporated per reaction mixture. 



TABLE 8 



EFFECT OF DELAYED ADDITION OF NTPs ON 
POLYADENYLIC ACID SYNTHESIS 



A standard reaction mixture containing 0.5 ymoles MnCl2r 

0.037 A^,„ denatured DNA, 5 pg enzyme and NTPs as indicated 
260 ^ ^ 

were used. Delayed addition of the NTPs was at times in- 
dicated during a standard 2 min. incubation. NTPs con- 
tained GTP , CTP and UTP . 



IITPs Added 



3. 

AMP Incorporated 
pmoles % 



None 16 40 

0.25 umoles each, min. 400 100 
0.0025 pmoles each, 

min. 52 13 

1 min. 60 15 
5 min. 88 22 



^Total pinoles incorporated per reaction mixture. 



69 



•consistent with the synthesis of poly (A) before NTP addition. 
Enzyme 

Enzyme was required for AMP incorporation and 
increased as a linear function of the enzyme protein present 
(Figure 2 0) . Replacement of denatured DNA with native DNA 
caused greater than a 90 percent decrease in AMP incorpora- 
tion. Therefore, the synthesis of poly (A) was directly 
proportional to the enzyme level and the enzyme preferred 
denatured DNA. 

' Product Characterization 

The acid-insoluble product synthesized with native 

2+ 2+ 

or denatured DNA with either Mg or Mn as cofactor 
remained insoluble after heating at 10 0° in 1 percent SDS 
for 5 min. (Table 9). As anticipated, if poly (A) longer 
than 12 nucleotides was accumulated in the reaction with 
denatured DNA, a more acid-insoluble product was synthesized 
and it too remained acid-insoluble after treatment. 

Nearest neighbor frequency analysis of products 
' synthesized from [a- P]ATP in the. absence of the NTPs 
resulted in a 0.90 ApA frequency (Table 10), indicating 
poly (A) synthesis. Incorporation into CpA, GpA and UpA 
accounted for 10 percent of the label incorporated. 

The average chain length of the product was estimated 
by the ratio of AMP to adenosine after alkaline hydrolysis. 
The chromatographed AMP fraction contained 2,200 cpm. 



70 




PR0TEir4 pq 



Figure 20. POLYADEI^TYLIC ACID SYNTHESIS AS A •, 
FUNCTION OF ENZYME CONCENTRATION 

Assayed in a standard reaction mixture containing 
0.5 yraole MnCl2, 0.37 ^^SO either native 

(solid circles) or denatured (solid triangles) , 
and enzyme protein as indicated. 



71 



TABLE 9 

EFFECT OF HEATING AND SDS ON 
RNA POLYMERASE PRODUCT 



Standard reaction mixture with 0.37 A„,„ DNA, and either 

260 

1.0 ymoles MgCl2 or 0.4 ymoles MnCl2 as indicated. Heating 
was for 5 min. at 100°. SDS was 1% where indicated. Each 
assay contained 6.5 ug enzyme. 



Components in Reaction Mixture AMP Incorporated (pmoles)* 

Template Metal Control Heated Heated in SDS 



Native 


Mg . 


660 


610 


625 


Native 


Mn 


627 


632 


630 


Denatured 


Mg 


805 


800 


840 


Denatured 


Mn 


1160 


1160 


1190 



Total radioactive nucleotide incorporated per reaction 
mixture . 



72 



TABLE 10 

NEAREST NEIGHBOR FREQUENCY ANALYSIS OF 
POLYADENYLIC ACID PRODUCT 



A standard reaction mixture containing 0.37 A^^„ denatured 

260 

DNA, 0.10 ymoles [a-32p]ATP (specific activity 30 mCi/mmole) , 
0.4 ymoles MnCl2 and 10 yg enzyme was incubated at 30° for 
60 min. 1% SDS was added to the reaction, it was passed 
over sephadex G25, material in the excluded volume was 
hydrolyzed in 0.3 M KOH for 18 h at 37°, adsorbed to acti- 
vated charcoal and the eluted nucleotides lyophilized, re- 
suspended in H^O and resolved by paper electrophoresis (see 
METHODS) , • . 



CPM 

CpA. ApA GpA UpA 

Added Recovered 



11,000 4,500 ■ 0.01 0.90 0.04 0.05 

^Some of the sample was lost during lyophilization . 



73 



TABLE 11 ' 
AVERAGE CHAIN LENGTH OF POLYADENYLIC ACID 



A standard reaction mixture was used containing 0.037 A^,„ 

250 

denatured DNA^ 0.5 ymoles MnCl2/ no NTPs and 12 ug KNA 
pol3^erase. The reaction was incubated for 60 min. at 30°. 
Yeast RNA (1 mg) was added and the product precipitated by 
the addition of 0.3 ml of cold 1.0 M HCIO^ containing 5 mM 
sodium pyrophosphate. The ppt. was washed 4X with 1 ml of 
cold 0.5 M HCIO^ and then alkaline hydrolyzed and 
electrophoresised (see METHODS) . 



Compound CPM 



AMP 
Adenosine 



2,200 

<22 



74 

whereas the radioactivity detected in the adenosine fraction 
did not exceed background (Table 11) . The sensitivity of 
the assay for adenosine was ±22 cpm; therefore the lower 
estimate of the average chain length for poly (A) was 2,200/ 
22, or greater than 100 nucleotides per chain after 60 min. 
at 30° . ' ■ . 

Suininary 

Maize UNA polymerase synthesized polyadenylic acid in 

the presence of denatured DNA, ATP and metal, preferably 
2+ 

Mn . There was greater than a 17-fold preference for 
ATP over UTP as substrate for homopolymer synthesis. Low 
levels of the NTPs as a mixture or added individually in- 
hibited poly (A) synthesis, independent of the time of 
addition. Poly (A) synthesis was directly proportional to 
the amount of enzyme protein added. The product remained 
acid-insoluble after boiling withSDS. The average chain 
length of the product accumulated after 60 min. incubation 
was greater than 10 nucleotides and the ApA frequency was 
0.90. 

Comparison of RNA and Polyadenylic 
Acid Synthesis 

The two AMP incorporating activities measuring RNA 
and poly (A) synthesis were apparently catalyzed by the same 
enzynie; RNA polymerase II (69) . The evidence for this 



75 



conclusion was indirect, since the enzyme preparation was not 
homogeneous. The data presented thus far in support of one 
enzyme catalyzing both syntheses includes: 1) Both activ- 
ities were eluted simultaneously from DEAE-cellulose by 
an (NH^)2S0^ gradient with a constant RNA and poly (A) activity' 
ratio in the active fractions. 2) Both activities required 

DNA for AMP incorporation. 3) Both activities had the 
2+ 2+ 

same Mg and Mn cof actor optima. 4) The for ATP was 
the same with native and denatured DNA. Additional com- 
parisons of RNA and poly (A) synthesis were made to further 
associate the accumulation of both products with one 
enzyme. These included inhibitor activities, nuclease 
sensitivity of products as a function of time and template 
specificity. 

Effect of Inhibitors 

Three types of inhibitors of RNA polymerase were 
investigated, those that bind specifically to the enzyme 
protein such as a-amanitin and rifampicin; those that 
bind to the template such as actinomycin D; and those 
that compete with the substrate such as cordycepin. The 
inhibitors were tested both in the presence and absence 
of the NTPs, therefore measuring the sensitivity of both 
RNA and poly (A) syntheses. 

Low concentrations of a-amanitin inhibited both RNA 
and poly (A) activity equally (Table 12). A titration of 
RNA polymerase activity with increasing levels of a-amanitin 



76 



TABLE 12 

INHIBITOR SENSITIVITY OF MAIZE RNA POLYMERASE 



A standard reaction mixture contained 0.037 A„,„ denatured 

2 6 

DNA, 0.5 umoles MnCl2, 0.05 ymoles ATP, 5 yg enzyme and in- 
hibitors as indicated added at zero times. 



Inhibitor Concentration Percent of Control 



Plus NTPs^ Less NTPs^ 



a-amanitin 


0.1 yM 


6 


7 




1.0 yM 


1 


2 


Actinomycin D 


7.0 yM 


14 


128 




70.0 yM 


1 


87 


Cordycepin 


0.13 mM 


100 


101 




0.26 mM 




100 


Rif ampicin 


50.0 yg/ml 


100 


110 



336 pmoles AMP incorporated equaled 100%. 
138 pmoles AMP incorporated equaled 10 0%. 



77 



"indicated 50 percent inhibition at 1 x 10 M (Figure 21). 
Since a-amanitin is specific for the type II eukaryotic 
RNA polymerase (32) , this inhibition of activity indi- 
cated that the maize enzyme was a type II RNA polymerase 
and that it catalyzed both RNA and poly (A) synthesis (69). 

Actinomycin D inhibited AMP incorporation in the 
presence of the NTPs (Table 12) . However, in the absence 
of the NTPs, AMP incorporation was stimulated. Even at 
very high actinomycin D concentrations (70 yM) , there was 
only a slight inhibition of poly (A) synthesis. Therefore, 
the sensitivity of the RNA polymerase to actinomycin D was 
dependent upon the presence of the NTPs. In the presence 
of the NTPs, AMP incorporation was inhibited 50 percent 
at 2 yM actinomycin D and was inhibited 95 percent at 50 yM 
(Figure 22) . 

The inhibition by a-amanitin and the resistance to 
cordycepin and rifampicin excluded the presence of two 
potential enzyme contaminants (Table 12). The maize NTP : 
exotransf erase is resistant to a-amanitin and sensitive 
to cordycepin while bacterial RNA polymerases are resistant 
to. a-amanitin and sensitive to rifampicin. Therefore the 
presence of ISITP : exotransf erase activity from maize tis- 
sue and bacterial RNA polymerase activity from contaminat- 
ing bacteria were ruled out. 

Utilizing the resistance of purine-purine 
phosphodiester linkages to pancreatic ribonuclease (54) , 



78 



IQOp 




ALPHA AyANITIM X\0 U 



Figure 21. ALPHA-AMANITIN TITRATION OF RNA POLYMERASE 



Assayed in a standard reaction mixture containing 1.0 ymole 
MgCl2f 0.37 ^2e0 ^^'^^^^ D'NA, 18 yg RNA polymerase and 
a-amanitin as indicated. Inhibitor was added just prior to 
addition of enzyme to the reaction mixture. 




ACTIMOMYCIN. D 



Figure 22. ACTINOMYCIN D TITRATION OF RNA POLYMERASE 

Assayed in a standard reaction mixture containing 1.0 ymole 
MgCl-, 0.37 A^gQ native DNA, 18 yg RNA polymerase and 
actinomycin D as indicated. Inhibitor was added just prior 
to addition of enzyme to the reaction mixture. 



80 

we determined the amount of poly (A) accumulated with dena- 
tured DNA in a standard reaction mixture as a function of 
time. AMP incorporation in the absence of the NTPs was 
not affected by the ribonuclease treatment (Figure 23, 
panel B) . In data not shown here AMP incorporation in a 
standard reaction mixture containing denatured DNA and 
4 yg ribonuclease showed no inhibition of incorporation, 
consistent with the exclusive synthesis of poly (A) . In 
the presence of the NTPs, there was a decrease in acid- 
insoluble AMP when the product was treated with ribonuclease 
(Figure 23, panel A). However, the 10-min. product was 
/jQnly 10 percent sensitive to treatment whereas the 60-min. 
product was 45 percent sensitive. This result suggests 
early synthesis of poly (A) and later synthesis of RNA. 

Template Requirement 

Both calf thymus and maize DNA supported RNA and 

poly(A) synthesis (Table 13). In the absence of the NTPs, 

where only poly (A) was synthesized, denatured maize and 

calf thymus DNA supported equal AMP incorporation. Since 

poly (A) synthesis was supported by DNA from both species, 

the synthesis of poly (A) was not an artifact of the 

heterologous system (calf thymus DNA and maize polymerase) . 

The lower AMP incorporation in the complete system 

observed on denatured maize DNA as compared with denatured 

calf DNA reflects the presence of native DNA in the heated 

maize preparation; note the low hyperchromicity of the 
maize DNA. 




81 



S8|0iLid Q3l¥?^0d.H0DNI 



E 



Ui 
F^ 
O 

a 
o 
Pi 

fu 

EH 

W 
Q 

H 
ft 
H 
Q 
I 

< 

Q 

Q 

Pi 
D 
Eh 
< 

M 
Q 

O 

>^ 
EH 
H 
> 
H 
EH 
H 
CQ 

Cfl 

H 
CO 
>< 
H 
1^ 
U 
tD 

o 
m 

H 



CO 

0) 

u 

-H 



0) 

H 03 
O 

g 

-P 

in 
• Q) 

O 4:: 
+J 

13 

-P 

< 

Q 



g 
(0 



o 

H 

(U +J CQ 
E CD 

rH u ■ri 

•P (D 

O -P 

M (0 

O CU 

H U 

Xi -P 
O 

■H 0) 

(1) o\o cc; 

OJ c — 

>i Q 

nj rH O 

c o Q) m 

CI.-P &H 

<; 4J s 

CN -H 0) 

i< tn o cn 

3- 0) — 

ro LD 0j 0) 



CU 
U 

-P 



Id 



o 



S-i ftj 



■H 

■rH 



.CJ 



•p 

-•-H Hi 

cn a) a 
CM o 
tti Eh 13 ^ 

-p s c: -H 

C (CM 
O -P 

O 13 o 

O (U 
CU 4:3 > -P 



U -P 

-p ^ 

-H CQ 



M O 
(D fO 

!h a, 



=1. 



c 0) CU 
c & 
to 

!h • W 

(U m ^ o (U 

M a -P rH 

Eh X ^ U 

s -H -p 



o 

•H 

o 

(0 



t3 4-) 

c: -H 
-p 



-H 

O t3 



-p -p 



(U (U 



tfl i< O fO o 

CU 



(T3 rH 
0) 



O X! 
fd -H 13 



CD 



•H 

CU 

>1 CNU 

(d rH -H T3 -H 

m u ^ -H 

cn a c 

<; s -H 



0) 

-P !h +J 

d o fU 
o 



o a 



82 



TABLE 13 

COMPARISON OF CALF THYMUS AND MAIZE DNA AS 
TEMPLATES FOR RNA POLYMERASE 



Assayed in a standard reaction mixture containing 0.5 ymoles 
MnCl2/ 0.037 A^gQ DNA and 6.4 yg RNA polymerase 

■ ■ AMP Incorporated^ (pmoles) 
DNA 

Complete Less NTPs 

Native Calf Thymus 
Native Maize 

Denatured Calf Thymus 
. c 

Denatured Maxze 



^Total pmoles incorporated per reaction mixture. 

Hyperchromicity 26%. 
^Hyperchromicity 11%. 



219 
260 

400 
300 



29 

46 

160 
163 



83 



If RNA polymerase catalyzed two reactions, RNA 
synthesis and poly (A) synthesis, and if both reactions 
required a template, then no AMP incorporation on 
polyCdAT), the alternating copolymer, would be expected 
in the absence of UTP . As seen in Table 14, AMP incorpo- 
ration on poly(dAT) was observed only in the presence of UTP. 

Poly(dAdT), the complementary homopolymers , were 
also templates' for AMP and UMP incorporation. AMP incor- 
poration was stimulated 2-fold by the addition of UTP/ 
suggesting synthesis of poly(U) on a poly(dA) template. 

However, when UMP incorporation was measured directly 
14 

(with C-UTP) little product accum^ulated unless ATP was 
added to the reaction mixture, suggesting that poly (A) 
synthesis was required for UMP incorporation. Incorporation 
with the complementary homopolymers was sensitive to 
a-amanitin indicating that the RNA polymerase was catalyzing 
the ATP stimulated UMP incorporation. 

Sxlmmary 

RNA and poly (A) synthesis by RNA polymerase II was 
confirmed by the sensitivity of both to a-amanitin, Ribo- 
nuclease resistance suggested that the early product was 
poly (A) . Requirements for poly (A) and RNA synthesis with 
several deoxyoligomers demonstrated that template was 
required for both poly (A) and RNA synthesis. The 
differential "sejisitivity to actinomycin D indicated that 



84 



* TABLE 14 

RNA POLYMERASE ACTIVITY WITH SYNTHETIC TEMPLATES 



Enzyme activity assayed in a standard reaction mixture con- 
taining 0.5 uinoles MgCl2, 0. 037 ^260 ^i"*^^^^ 0-1 umole 
labeled ATP or 0.05 ymole labeled DTP and 4 vg RNA poly- 
mexase. 



Nucleic Acid Substrate , Incorporation (pmolesf 

AMP UMP 



Poly (dAT) 


14 

C-ATP 






13 




Poly (dAT) 


14 

C-ATP 


+ 


UTP 


1,090 




Poly(dAdT) 


C-ATP 






104 




Poly (dAdT) 


14 

C-ATP 


+ 


UTP 


211 




Poly (dAdT) 


14 

C-UTP 








4 


Poly (dAdT) 


14 

C-UTP 


+ 


ATP 




40 


Poly (dAdT) 


14 

C-UTP 


+ 


Id 

ATP (a-aman) 




0.2 



Total radioactive nucleotide incorporated per reaction 
mixture . 

a-amanitin added to 20 ug/ml. 



the polymerase utilized different sequences in the DNA 
but not necessarily different DNA molecules for KNA and 
poly (A) synthesis. 



DISCUSSION 



Enzyme Preparation 

St-udies of both product characterization and the 
initiation of transcription required an enzyme of a much 
higher specific activity than the soluble maize RNA poly- 
merase purified by Stout and Mans (27) . In addition, the 
net yield of active polymerase had to be increased and the 
purification procedure made more convenient and rapid for 
.'effective experimental progress. These goals were achieved. 
The RNA polymerase specific activity was increased 10-fold, 
the net yield was more than doubled, and the time required 
for each preparation v/as reduced from 7 days to 10 hours. 
These ixRprovements resulted from 4 major alterations in the 
procedure of Stout and Mans (27) , including changes in: 
1. the storage of the starting material; 2. the homogeniza- 
tion procedure; 3. the salt equilibration of the soluble 
proteins; 4. the DEAE-cellulose chromotography procedure. ■ 

Previously (27) , adequate grain was germinated under 
running water and/ after 5 days, the seedlings were harvested 
just before each enzyme preparation. In contrast, after 
large scale germination and harvest (1 to 3 kg) followed 
by storage of the seedlings at -76° (Revco freezer) in 

86 



87 



alximinum packets, each enzyme preparation required only the 
removal of a weighed packet of tissue from the freezer just 
before homogenization. The specific activities of the 
homogenates from freshly harvested tissue and from tissue 
stored at -76° were identical. 

In the original procedure (27) , the shoots and roots 
were pulverized under liquid nitrogen and then, in small 
batches, passed through a French pressure cell. This was 
replaced by rapid homogenization, in one batch, in a 
Waring blender. During homogenization, the enzyme was 
protected from oxidation (foaming) with 50 mM 2-mercapto- 
ethanol. The enzyme specific activity of the blender 
treated material (Table 1) was identical to material from 
the French pressure cell (27) . 

The removal of (NH^)2S0^ from the salt-precipitated 
enzyme fraction by a 4 h dialysis against Buffer R (27) , 
was replaced by a 20 min. gel filtration procedure. The 
Sephadex G50 was equilibrated and the proteins eluted in 
-1;he excluded volume with 0.2 M (NH^)2S0^. At this salt 
concentration all RNA polymerase was bound to the DEAE- 
cellulose column, but only a small fraction of the total 
protein was bound (illustrated by A2gQ in Figure 3) . 

Previously (27) , RNA polymerase was eluted with a 
shallow Tris-HCl gradient (500 ml, 0.05 to 1.0 M). This was 
replaced by a steep (NH^) 250^ gradient (60 ml, 0.2 to 1,0 M) 
that eluted concentrated, highly active, RNA polymerase 



88 



(Table 1) . Stout and Mans (27) had concentrated the dilute 
Tris-HCl eluted enzyme by salt precipitation. Because the 
^ (Wd^) 2^^)^ eluted enzyme was concentrated, the salt precipita- 
•tim was obviated, and the 70 to 90 percent loss of activity 
such precipitation, caused, was avoided. In addition to 
the removal of most of the contaminating proteins, most, if 
not all, nucleic acids were removed. The ^250^'^280 '^^ 
material added to the column was 10, whereas the -^260'^'^280 
of the eluted fractions containing polymerase was 1.5 to 
2.0. Removal of nucleic acids during DEAE-cellulose 
chromotography apparently decreased the stability of the 

polymerase. The polymerase , was stable to salt precipi- 
tation before, but not after, chromotography. Addition of 

■ DNA, to the eluted fractions containing the polymerase, 

■ prior to precipitation with (NH^)2S0^ improved the recovery 
of polymerase activity (Table 3) , supporting the premise 
that the polymerase requires nucleic acid for stability. 

The recovery of active enzyme free of nucleic acid 
« fflay, therefore, result from the short time between elution 
from DEAE-cellulose and storage in liquid nitrogen. During 
the absence of nucleic acid, the RNA polymerase complex 
may either aggregate or disassociate, but in either case 
it becomes inactive. This hypothesis was supported by the 
polyacTylamide gel electrophoresis data (Figure 5) . The 
presence of one dark band and multiple smaller bands might 
represent subunits which have aggregated and/or disassociated 
from the polymerase complex. . 



89 



Strain et al. (47) reported the partial resolution 
of two type II maize RNA polymerases during DEAE-cellulose 
chromotography; one which preferred native DNA and one 
which preferred denatured DNA. The conditions of purifi- 
cation resulted in very low specific activities, perhaps 
reflecting an advanced state of polymerase degeneration. 
The possible degeneration of RNA polymerase is supported 
by the data of Chambon (39) with the detection of RNA poly- 
merase protein subunits of decreasing size, designated Bl 
and B2. The partial separation of two RNA polymerase 
activities by Strain et al. (47) would be consistent with 
the presence of one less degenerate RNA polymerase, and 
one in an advanced state of degeneration. 

Enzyme Characterization 

Maize RNA polymerase resembles the type II eukaryotic 
RNA polymerases (1) . The position of the maize RNA poly- 
merase in the DEAE-cellulose elution profile at 0.35 M 
(NH^)2S0^ (Figure 4) was as expected for a type II RNA 
polymerase (32). Like the other type II enzyme (32), maize 
RNA polymerase can utilize both native and denatured DNA 

as a template (Table 4); it requires high salt (Figure 14) 
2+ 2+ 

and exther Mg of Mn as metal cof actors (Table 4) . The 

2+ 2+ 

strong preference shown for Mn over Mg by the sea urchin. 



90 



rat and calf thymus enzymes (32) was not observed for 

maize RNA polymerase (Table 4) . Rather than the 5-to 

14-fold preference expected, the maize enzyme exhibited, 

2+ 

at most, a 2-fold preference for Mn . This low ratio 
of Mn-dependent to Mg-dependent activity could be a unique 
property of maize RNA polymerase, or it could reflect a 
variable rate of polymerase degeneration. If the Mg- 
dependent activity was more labile than the Mn-dependent 
activity, then the low Mn/Mg activity ratio may have 
resulted from the rapid purification procedure which per- 
mitted the maize Mg-dependent activity to remain active. 
All the type II RNA polymerases have the unique property 
of a-amanitin sensitivity (32) . The evidence involving 
enzyme purification and assay characteristics suggested, 
and the inhibition by a-amanitin (Figure 21) confirmed 
the inference, that the maize enzyme was a type II poly- 
merase (45). More importantly, a-amanitin inhibition 
indicated poly (A) synthesis was also catalyzed by the 
RNA polymerase II (Table 12) . The synthesis of both RNA 
and poly (A) by the same enzyme was supported by the co- 
purification of both activities during DElAE-cellulose 
chromotography , and the identical metal optima and the 

identical K for ATP. Therefore, the same enzyme was 
m 

responsible for both activities. 



91 

RNA and poly (A) synthesis by the same enzyme could ' 
be distinguished by alterations in the reaction mixture 
components since: 1. ATP was a substrate for both RNA 
and homopolymer synthesis, while UTP was a substrate only 
for RNA synthesis (Table 6). 2. Native DNA and denatured 
DNA (Table 4), poly(dAT) and poly(dAdT) (Table 14), all 
satisfied the template requirement for RNA synthesis, 
but only denatured DNA and poly (dAdT) satisfied the tem- 
plate requirement for poly (A) synthesis. 3. The NTPs 
were required for RNA synthesis (Figure 11), while poly (A) 
synthesis was inhibited by low NTP concentrations (Fig- 
ure 19). 4. Actinomycin D inhibited RNA synthesis but 
stimulated poly (A) synthesis (Table 12). 

The synthesis of poly (A) with ATP, but not of 
poly (O) with UTP, indicated the RNA polymerase synthesized 
only one homopolymer. Synthesis of homopolymer required 
nucleic acid (Table 4) , and the nucleic acid was utilized 
as a template and not as a primer (Table 14) . Therefore, 
poly(dT) regions must serve as templates to synthesize 
poly (A) . Since Watson-Crick base pairing would require 
a poly(dT) template for poly (A) synthesis on DNA, as well 
as on synthetic oligomers, DNA denaturation must increase 
the exposed poly(dT) regions and provide the required 
template for poly (A) synthesis. 



Poly (A) and SNA are probably synthesized on the 
same template. Addition of the NTPs increased AMP and 
DMP incorporation equsilly (Table 6) , reflecting RNA 
synthesis. The increased AMP incorporation occurred above 
the level of homopolyiBEer synthesis measured when the NTPs 
were absent. However if very low. levels of NTPs were 
added, poly (A) synthesis was inhibited (Figure 19). This 
inhibition was identical to the NTP inhibition of E. cOli 
PtNA polymerase during poly (A) synthesis by reiterative 
transcription (46) . Ttie E. coli RNA polymerase was in- 
hibited when the enzyme began transcribing RNA with an 
insufficient concentraiiion of NTPs to support measurable 
UNA synthesis. This resulted in a net inhibition of 
AMP incorporation. Since delayed addition of low level 
NTPs inhibited maize poly (A) and RNA synthesis, the syn- . 
thesis of poly (A) aiid RNA probably occurred on the same 
DNA molecule. Utilizat;ion of the same DNA molecule for 
poly (A) and RNA synthesis was further supported by the 
actinomycin D resistance of poly (A) synthesis. In the 
absence of the NTPs, poly (A) synthesis was refractory 
to actinomycin D, but when NTPs were added, all incorpo- 
ration was inhibited by the actinomycin D (Table 12) . 
Since actinoirrycin D preferentially binds guanosine and 
inhibits RNA synthesis by impeding the progress of the 
enz^^e along the DMA template (70), the poly (dT) -rich 
regions of the DNA^ unable to bind actinomycin D, would 



93 



remain available for reiterative poly (A) synthesis. With 
NTP addition, reiterative transcription would cease, RNA 
synthesis would begin, and the enzyme would encounter an 
actinomycin D-guanosine complex and be inhibited. There- 
fore, both the NTP inhibition and actinomycin D inhibition 
data are consistent with poly (A) and UNA synthesis occurring 
on adjacent regions of the same DNA template. 

Poly (A) and RNA are synthesized by the maize. RNA 
polymerase II, and may be attached to one another. Maize 
RNA polymerase synthesized RNA with native and with de- 
natured DNA, but the ApA frequency went from 30 percent 
with native DNA (27) to 44 percent with denatured DNA 
(Table 5) . The increase in ApA frequency was not neces- 
sarily a change in the base composition of the RNA syn- 
thesized, but may have resulted from the simultaneous 
synthesis of poly (A) and RNA on the denatured DNA template. 
The early product synthesized in a complete reaction 
mixture containing denatured DNA was resistant to 
pancreatic ribonuclease , while late product was more 
sensitive (Figure 23) . This suggested the initial product 
contained poly (A) and the subsequent product contained 
RNA. Early poly (A) synthesis was also suggested by the 
kinetics of AMP incorporation: a rapid initial rate in 
which apparently all the nucleotides incorporated were 
labeled; and a subsequent lower rate in which approximately 
30 percent of the incorporated nucleotides were labeled 



94 



(Figure 36) . Therefore, the rate of nucleotide elongation 
appeared to be constant throughout poly (A) and RNA syn- 
thesis, although the rate of AMP incorporation changed. 
If poly (A) synthesis precectes rna synthesis, then poly (A) 
would logically be expected at the 5' end of the RNA, 
since the known RNA polymerases synthesize product from 
the 5' to the 3' end (15). The linkage of poly (A) and 
RNA, suggested by the data from maize, is strikingly 
similar to that obtained with vaccinia virus cores by 
Kates (53). Poly (A) synthesized by vaccinia cores 
apparently was attached to RNA, but only when both were 
synthesized simultaneously. With ATP alone, poly (A) , 
ISO nucleotides long, was synthesized and not covalently 
attached to RNA synthesized after the addition of the 
NTPs. If both ATP and NTPs were initially present, poly (A) , 
50 nucleotides long, was synthesized and it was apparently 
attached to the RNA (53). The data on maize poly (A) syn- 
thesis are consistent with the vaccinia system, therefore 
long poly (A) sequences may be synthesized in the absence 
of the NTPs, while short poly (A) sequences, attached to 
RNA, may be synthesized in the presence of the NTPs. 
Although resistance of products to RNase digestion resulting 
from protein-product and DNA-product complexes (44) has 
been ruled out in subsequent experiments, the attachment 
of poly (A) to RNA has not been unequivocally demonstrated 
in either the maize RNA polymerase reaction or the vaccinia 
core reaction. 



95 



Poly (A)' Synthesis and the Initiation 
of Transcription 

A major unsolved problem in eukaryotic transcription 
is the initiation of RNA synthesis, including the recogni- 
tion of the binding site on DNA and selection of the 
proper strand for transcription.. The discovery of poly (A) 
synthesis by maize RNA polymerase II indicated the 
phenomenon of reiterative poly (A) synthesis by prokaryotes 
(E. coli ) could be extended to eukaryotes . Unlike the 
prokaryotes, maize RNA polymerase II synthesizes poly (A) 
and RNA during the same reaction. In prokaryotes, ATP 
and GTP are found at the 5' terminus of newly synthesized 
RI'IA. GTP in: t"' at ion maybe an artifact of the in vitro 
system (71) , suggesting ATP is the first base incorporated 
during initiation of transcription. Perhaps it is the 
beginning of a short poly (A) strand, or perhaps the remnant 
of a longer discontinuous poly (A) strand. 

Transcription of native DNA is asymmetric (72) , that 
is, only one strand of the DNA at any one region of the 
DNA is transcribed into RNA. If the RNA polymerase had 
a binding affinity for only poly (dT) -rich regions on one 
strand of the double helix, the complementary strand con- 
taining poly (dA) would not be bound. Therefore, not only 
selection of the proper site, but selection of the proper 
strand of DNA, might be attributed to poly (A) synthesis. 



96 



Jt. i,s interesting to note that poly (A) is resistant 
to iritBantit^.aase digestion. If poly (A) were involved in 
the initiation of transcription, its metabolic stability 
would insure transcription by forming a nuclease resistant 
.'initiation complex. Perhaps the reason that the denatured 
DNA out-competed native DNA for the maize RNA polymerase 
(Figure 7) was that the maize polymerase had a greater 
affinity for the exposed poly(dT) regions. Since poly(dT)- 
'rich regions would be most easily denatured (A:T pairs 
are less strongly hydrogen bonded than G:C pairs) , the 
maize RNA polymerase would bind to the more accessible 
regions of the partially denatured DNA. These RNA poly- 
merase binding sites could arise in vivo via the unwinding 
proteins isolated by Alberts and Frey (73) . 

Both poly (A) and RNA are synthesized by the maize 
RNA polymerase II and by E. coli RNA polymerase. Since 
both enzymes can utilize a duplex DNA template, and since 
eukaryotic RNA polymerase may have a subunit structure 
analogous to the paired structure of E. coli RNA polymerase 
^■2'^'^') (see reference 11 and 38), there may be two 
active sites (a, 3) per RNA polymerase complex. One site 
may recognize a binding site on one strand of the DNA tem- 
plate and synthesize poly (A) and RNA on that strand, while 
the other site idles. Should the polymerase encounter an 
initiation site on the opposite strand,, one site synthesizes 
poly (A) and the other site the RNA, but sequentially and 



97 



in opposite directions. In APPENDIX B, a diagramatic 
.inodel;.,,fo|: the initiation of transcription is presented. 
This model includes the initiation of RNA synthesis by 
poly (A) , the synthesis of RNA on either the same or 
opposite DNA strands, and the presence of an RNA poly- 
merase containing two active sites, each site capable 
of binding DNA and of polymerizing nucleotides. 

The partially resolved peaks of two maize RNA 
•polymerase II activities reported by Strain et al. (47) 
might reflect a degenerate RNA polymerase that can syn- 
thesize only RNA, and a less degenerate enzyme that can 
syhthes'izfe both poly (A) and RNA. The apparent preference 
of one RNA polymarase II for denatured DNA may represent 
the synthesis of poly (A) by the less degenerate enzyme. 
Similarily, the failure to detect homopolymer synthesis 
by Stout and Mans (44) , may reflect a similar degeneration 
of the in vivo RNA polymerase complex. Maize RNA poly- 
merase purified by the procedure described in METHODS 
does retain the ability to synthesize both poly (A). This 
rapid purification procedure may be necessary to prevent 
the degeneration of the RNA polymerase complex with the 
associated decrease in activity, perhaps including the 
loss of poly (A) synthesis. Further purification of the 
maize RNA polymerase will determine whether such degenera- 
tion takes place, and whether the two polymerase types can 



98 

be detected; those that can synthesize only RNA and those 
that can synthesize both poly (A) and RNA. 



CONCLUSION 



Maize DNA-dependent RNA polymerase II synthesizes 
both RNA and polyadenylic acid utilizing a DNA template. 
The evidence is consistent with a model where polyadenylic 
acid synthesis precedes the synthesis of RNA in the same 
ensyme-template complex during the initiation of 
transcription. 



99 



100 



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2 . Silvestri , L . (ed . ) . Lepetit Colloquia on Biol . and 

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3. Hanly, E. W. (ed.) . RNA in Development , The Park City 

Inter. Symp . on Prob . in Bio . (1969) , Univ. of Utah 
Press, Salt Lake City, Utah. 

4. Burgess, R. R. (1971). Ann . Rev . Biochem. , £0:711. 

5. Von Hippel, P. H., and J. D. McChee (1972). Ann . Rev . 

Biochem . , 41:231 

6. Losick, R. (1972). Ann . Rev. Biochem . , 41:409. 

7. Zubay, G. L. (ed.). Papers in -Biochemical Genetics , 

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N. Y. 

8. Watson., J. D. MoleGju].ar Bio_logv of the Gene., 2nu ed. 

(1970). W. a7" Benjamin, '~Inc'. New" York," N 

9. Mahler, H. R. and Cordes, E. H. Biological Chemistry 

(1966) . Harper and Row, New York, N.Y. 

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APPENDIX A 



" Incorporation and ATP Specific Activity 

All activity measurements were based on the 
incorporation of radioactivity into an acid-insoluble 
product. Since the products formed by the RNA polymerase 
contained AMP [8--^^C]ATP was used as the labeled precursor. 
The fraction of the product AMP labeled should, therefore, 
■i>e proportional to the ATP specific activity. To test this 
assumption, the specific activity of the ATP was varied 
from 2.2 to 6.5 yCi/ymole, at three different ATP concen- 
tra:tions: 0.25 mM, 0.5 mM and 1.0 mM. The amount of 
acid-insolub-le r.^d in activity synthesized in a standard 
reaction mixture was found to be directly proportional to 
the ATP specific activity (Figure 24) . The predicted re- 
lationship between AMP incorporation and the specific 
activity of the ATP substrate was, therefore, confirmed. 
Comparisons of enzymatic activities could now be made, 
even if the ATP specific activity was varied, provided- 
enzyme activity was expressed as pmoles of AMP incorporated. 

When a more radioactive product is needed, such as 
during product characterization and hybridization procedures, 
the cpm incorporated into the product can be increased 
with additional labeled ATP. This can be achieved by 
keeping the amount of labeled ATP constant and decreasing 

105 



j 



106 




ATP SPECIFIC ACTIVITY C/M 



Figure 24. AMP INCORPORATION AS A FUNCTION OF 
ATP SPECIFIC ACTIVITY 

Assayed in a standard reaction mixture containing 
1.0 ymole MgCl- / 0.37 A ^gg native DNA and 4 ug RNA 
polymerase. Tne ATP concentrations were as follows:. 
1.0 mM (solid circles), 0.5 mM (solid squares) and 
0.25 mM (solid triangles). 



107 



the amount of cold ATP added. The net effect is an 
increase in the specific activity of the labeled ATP. 
However, since the ATP concentration is no longer satu- 
rating the reaction, the total pmoles of Af4P incorporated 
decreases. This decrease in incorporation with lower ATP 
concentrations was a consistent percentage for all time 
points examined out to 40 min. (Figure 25) . If the ATP 
concentration was decreased from 1.0 mM to 0.5 mM, 86 per- 
cent of the maximum rate remained; if the ATP concentration 
was decreased to 0,25 mM, 68 percent of the maximum rate 
remained. Since the labeled ATP was held constant as the 
total ATP concentration was decreased there was a 2-fold 
increase, in specific activity with 0.5 mM ATP, and a 4-fold 
increase with 0.25 mM. The. combined result of the decreased 
amount of AMP incorporated and the increased labeling of 
that which was incorporated was a 72 percent increase in 
cpm for 0.5 mM ATP and a 164 percent increase in cpm for 
0.25 mM ATP. Therefore, the labeled substrate was conserved 
and more cpm were incorporated into the RNA polymerase 
product. 



108 



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APPENDIX B 



A Model for Polyadenylic Acid Initiation 
of Transcription 

Enzyme Structure 

The RNA polymerase consists of a core enzyme with 
paired subunits {a 2; ^i) ' Additional factors or subunits 
may confer template specificity or additional activities 
upon this core polymerase. Each pair of subunits (a, 6) 
of the core enzyme contains the ability to bind DNA and 
to catalyze RNA synthesis, therefore, creating the 
potential for two active sites per RNA polymerase complex 
(Fxgure 26) . . 

Enzyme Activity 

Properties of Site 1 

Site 1 synthesizes RNA and will bind Randomly on 

2+ 2+ 

denatured DNA. It will utilize either Mg or Mn and 
it will incorporate all four NTPs. It is sensitive to 
a-amanitin and actinomycin D. It will actively catalyze 
RNA for more than 90 min. in vitro. 



109 



110 



■ / T T T T T 



/" site 
V ^""$i't'e 



X A A 




y / . A.a"a'A a 
^ //" Site ! 



4 \— 




N y T T T T T 

IE 




DMA 



5' 



3' 



Figure 26. A MODEL FOR POLYADENYLIC ACID 
INITIATION OF TRANSCRIPTION 



II. 



Polyadenylic acid and RNA transcribed 
from the same strand of DNA. 

Polyadenylic acid and RNA transcribed 
from opposite strands of DNA. 



Ill 



Properties of Site 2 

• -.■ jSiS''©-..^ ■.synthesizes poly (A) and RNA, depending upon 

the assay substrate and template. It binds selectively 

to poly(dT) regions of denatured DNA and when synthesizing 

2+ 

poly (A) has a strong preference for Mn as a cof actor. 

In the, presence of ATP it will reiteratively tran- 
scribe the short poly(dT) template and synthesize long 
poly (A) sequences. In the presence of the NTPs, it will 
synthesize a short poly (A) sequence and then begin 
transcribing RNA. At low NTP concentrations, site 2 
begins transcribing into the DNA and is inhibited. It 
is inhibited by a-amanitin, but resistant to actinomycin 
D wiien syiiLi'io3j.i.j.iig pOj.y vA) . 

This model for initiation of transcription on 
double stranded DNA is based upon data with denatured 
DNA. The presence of a mechanism for selectively de- 
naturing the initiation region must, therefore, be 
present in vivo . 

It is suggested the poly (dT) -rich template region 
of the denatured DNA may be in a circular helix, permit- 
ting reiterative transcription and poly (A) synthesis 
to occur. 



BIOGRAPHICAL SKETCH 



Robert Henry Benson legally began to exist on 
December 15, 1942 in Chicago, 111. He lived with his 
parents, Henry and Mary Anne Benson, and four brothers, 
Gary, John, Richard and Kennith, until graduation from 
Miami Jackson High School in 1961. He was forced to 
leave home to attend the University of Florida (1961-62, 
1964-67) and the U. S. Coast Guard Academy (1962-64), 
graduating from the University of Florida in 19 67 with a 
bachelor's degree in chemistry. From 19 67 to 1972, he 
was cared for by the N. I.H. through the Department of 
Imiriunology and Medical Microbiology. In the Summer of 
1S69 he. i-aet Rusty J. Mane who has kept him busy ever since 
During a period of sensual insanity in October 197 0, he 
and Barbara A. Mendheim v/ere married and they have one 
Dachshund, Bernardine, age 2. 



112 



I certify that I have read this study and that in 
my opinion it conforms to acceptable standards of scholarly 
presentation and is fully adequate, in scope and quality, 
as a dissertation for the degree of Doctor of Philosophy. 




'^^^^^^---2 Rusty Jjij^ans, Chairman 

Pr-qfessor of Biochemistry 



I certify that I have read this study and that in my 
opinion it conforms to acceptable standards of scholarly 
presentation and is fully adequate, in scope and quality, 
as a dissertation for the degree of Doctor of Philosophy. 





7x JfJUiA^ 

Daniel Billen 
Professor of Radiology 



I certify that I have read this study and that in my 
opinion it conforms to acceptable standards of scholarly 
presentation and is fully adequate, in scope and quality, 
as a dissertation for the degree of Doctor of Philosophy. 

ijsslin W. Cramer 
^Associate Professor of 
Pharmacology and Therapeutics 



I certify that I have read this study and that in my 
opinion it conforms to acceptable standards of scholarly 
presentation and is fully adequate, in scope and quality, 
as a dissertation for the degree,^ of Doctor of Philosophy. 




George E. Gif f ord --^ 
Professor of Immunology and 
Medical Microbiology 



I certify that I have ]:ead this study and th£i.t in my 
opinion it conforms to acceptable standards of scholarly 
presentation and is fully adequate, in scope and quality, 
as a dissertation forthe degree of Doctor of Philosophy. 

Ira G. Rosen 
Assistant Professor 
Immunology and Medical 
Microbiology 



This dissertation was submitted to the Dean of the 
College of Medicine and to the Graduate Council, and v/as 
accepted as partial fulfillment of the requirements for the 
degree of Doctor of Philosophy. 



December, 1972 




Dean, Graduate School 



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